Gene therapy constructs and methods of use

ABSTRACT

Provided herein are improved gene therapy vectors and methods of use, in some embodiments, comprising sequences for improved expression and cellular targeting of a therapeutic protein.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No.62/664,741, filed Apr. 30, 2018; U.S. Provisional Application No.62/688,640, filed Jun. 22, 2018; and U.S. Provisional Application No.62/744,068, filed Oct. 10, 2018, each of which application isincorporated herein by reference in its entirety.

BACKGROUND

Genetic disorders arise via heritable or de novo mutations occurring ingene coding regions of the genome. In some cases, such genetic disordersare treated by administration of a protein encoded by the gene mutatedin the individual having the genetic disorder. Such treatment haschallenges however, as administration of the protein does not alwaysresult in the protein reaching the organs, cells, or organelle where itis needed. Furthermore, this treatment also often requires biweeklyinfusions, which are not needed with gene therapy, where a singletreatment can offer lasting relief. Therefore, gene therapy has thepotential to offer improved results over currently available treatmentsfor genetic disorders.

SUMMARY

Provided herein are compositions and methods for treatment of geneticdisorders using gene therapy. Also provided herein are gene therapyvector components and methods to be used in gene therapy for improvingprotein expression and increasing cellular uptake or delivery andintracellular or sub-cellular targeting of therapeutic proteins providedby gene therapy vectors.

In certain aspects, there are provided gene therapy vectors, forexample, gene therapy vectors comprising a nucleic acid constructcomprising, in 5′ to 3′ order: (a) a translation initiation sequence,and (b) a nucleic acid sequence encoding a therapeutic protein. In someembodiments, the translation initiation sequence comprises a Kozaksequence. In some embodiments, the translation initiation sequence andthe nucleic acid sequence encoding the therapeutic protein may overlap,such that the last three nucleotides of the translation initiationsequence are also the start codon for the therapeutic protein. In someembodiments, the Kozak sequence comprises the sequence AX₁X₂ATGA (SEQ IDNO: 28), wherein each of X₁ and X₂ is any nucleotide. In someembodiments, X₁ comprises A. In some embodiments, X₂ comprises G. Insome embodiments, the Kozak sequence comprises a nucleic acid sequenceat least 85% identical to AAGATGA (SEQ ID NO: 29). In some embodiments,the Kozak sequence differs from the sequence of AAGATGA (SEQ ID NO: 29)by one or two nucleotides. In some embodiments, the Kozak sequencecomprises AAGATGA (SEQ ID NO: 29). In some embodiments the Kozaksequence comprises a nucleic acid sequence at least 85% identical toGCAAGATG (SEQ ID NO: 44), wherein the last three nucleotides (ATG) arealso the start codon for the therapeutic protein. In some embodimentsthe Kozak sequence differs from the sequence of GCAAGATG (SEQ ID NO: 44)by one or two nucleotides. In some embodiments, the Kozak sequencecomprises GCAAGATG (SEQ ID NO: 44). In some embodiments the Kozaksequence comprises a nucleic acid sequence at least 85% identical toCACCATG (SEQ ID NO: 47). In some embodiments the Kozak sequence differsfrom the sequence of CACCATG (SEQ ID NO: 47) by one or two nucleotides.In some embodiments, the Kozak sequence comprises CACCATG (SEQ ID NO:47). In some embodiments, the nucleic acid construct further comprises anucleic acid sequence encoding a signal peptide capable of increasingsecretion of the therapeutic protein as compared to the therapeuticprotein without the signal peptide. In some embodiments, the signalpeptide is selected from a binding immunoglobulin protein (BiP) signalpeptide and a Gaussia signal peptide. In some embodiments, the BiPsignal peptide comprises an amino acid sequence at least 90% identicalto an amino acid sequence selected from the group consisting of SEQ IDNOs: 13-17. In some embodiments, the signal peptide differs from asequence selected from the group consisting of SEQ ID Nos: 13-17 by 5 orfewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 amino acid. In someembodiments, the BiP signal peptide comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 13-17. In someembodiments, the Gaussia signal peptide comprises an amino acid sequenceat least 90% identical to SEQ ID NO: 32. In some embodiments, the signalpeptide differs from the sequence of SEQ ID NO: 32 by 5 or fewer, 4 orfewer, 3 or fewer, 2 or fewer, or 1 amino acid. In some embodiments, theGaussia signal peptide comprises an amino acid sequence of SEQ ID NO:32. In some embodiments, the nucleic acid construct further comprises aninternal ribosomal entry sequence (IRES). In some embodiments, the IRESis a cricket paralysis virus (CrPV) IRES. In some embodiments, the IREScomprises a nucleic acid sequence at least 90% identical to SEQ ID NO:12. In some embodiments, the IRES comprises SEQ ID NO: 12.

In additional aspects, there are provided gene therapy vectorscomprising a nucleic acid construct comprising, in 5′ to 3′ order: (a) anucleic acid sequence encoding a signal peptide, and (b) a nucleic acidsequence encoding a therapeutic protein, wherein the signal peptide iscapable of increasing secretion of the therapeutic protein as comparedto the therapeutic protein without the signal peptide. In someembodiments, the signal peptide is selected from a bindingimmunoglobulin protein (BiP) signal peptide and a Gaussia signalpeptide. In some embodiments, the BiP signal peptide comprises an aminoacid sequence at least 90% identical to an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 13-17. In some embodiments, thesignal peptide differs from a sequence selected from the groupconsisting of SEQ ID Nos: 13-17 by 5 or fewer, 4 or fewer, 3 or fewer, 2or fewer, or 1 amino acid. In some embodiments, the BiP signal peptidecomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 13-17. In some embodiments, the signal peptide comprises aGaussia signal peptide. In some embodiments, the Gaussia signal peptidecomprises an amino acid sequence at least 90% identical to SEQ ID NO:32. In some embodiments, the signal peptide differs from the sequence ofSEQ ID NO: 32 by 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1amino acid. In some embodiments, the Gaussia signal peptide comprisesSEQ ID NO: 32. In some embodiments, the nucleic acid construct furthercomprises a translation initiation sequence. In some embodiments, thetranslation initiation sequence comprises a Kozak sequence comprisingAX₁X₂ATGA (SEQ ID NO: 28), wherein each of X₁ and X₂ is any nucleotide.In some embodiments, X₁ comprises A. In some embodiments, X₂ comprisesG. In some embodiments, the Kozak sequence comprises a nucleic acidsequence at least 85% identical to AAGATGA (SEQ ID NO: 29). In someembodiments, the Kozak sequence differs from the sequence of AAGATGA(SEQ ID NO: 29) by one or two nucleotides. In some embodiments, theKozak sequence comprises AAGATGA (SEQ ID NO: 29). In some embodimentsthe Kozak sequence comprises a nucleic acid sequence at least 85%identical to GCAAGATG (SEQ ID NO: 44). In some embodiments the Kozaksequence differs from the sequence of GCAAGATG (SEQ ID NO: 44) by one ortwo nucleotides. In some embodiments, the Kozak sequence comprisesGCAAGATG (SEQ ID NO: 44). In some embodiments the Kozak sequencecomprises a nucleic acid sequence at least 85% identical to CACCATG (SEQID NO: 47). In some embodiments the Kozak sequence differs from thesequence of CACCATG (SEQ ID NO: 47) by one or two nucleotides. In someembodiments, the Kozak sequence comprises CACCATG (SEQ ID NO: 47). Insome embodiments, the nucleic acid construct further comprises aninternal ribosomal entry sequence (IRES). In some embodiments, the IREScomprises an IRES selected from the group consisting of a cricketparalysis virus (CrPV) IRES, a picornavirus IRES, an Aphthovirus IRES, aKaposi's sarcoma-associated herpesvirus IRES, a Hepatitis A IRES, aHepatitis C IRES, a Pestivirus IRES, a Cripavirus IRES, a Rhopalosiphumpadi virus IRES, and a Merek's disease virus IRES. In some embodiments,the IRES comprises a nucleic acid sequence at least 90% identical to SEQID NO: 12. In some embodiments, the IRES comprises SEQ ID NO: 12.

In further aspects, there are provided gene therapy vectors comprising anucleic acid construct comprising, in 5′ to 3′ order: (a) an internalribosomal entry sequence (IRES), and (b) a nucleic acid sequenceencoding a therapeutic protein. In some embodiments, the IRES comprisesan IRES selected from the group consisting of a cricket paralysis virus(CrPV) IRES, a picornavirus IRES, an Aphthovirus IRES, a Kaposi'ssarcoma-associated herpesvirus IRES, a Hepatitis A IRES, a Hepatitis CIRES, a Pestivirus IRES, a Cripavirus IRES, a Rhopalosiphum padi virusIRES, and a Merek's disease virus IRES. In some embodiments, the IRES isa cricket paralysis virus (CrPV) IRES. In some embodiments, the IREScomprises a nucleic acid sequence at least 90% identical to SEQ ID NO:12. In some embodiments, the IRES comprises SEQ ID NO: 12. In someembodiments, the nucleic acid construct further comprises a translationinitiation sequence. In some embodiments, the translation initiationsequence comprises a Kozak sequence comprising AX₁X₂ATGA (SEQ ID NO:28), wherein each of X₁ and X₂ is any nucleotide. In some embodiments,X₁ comprises A. In some embodiments, X₂ comprises G. In someembodiments, the Kozak sequence comprises a nucleic acid sequence atleast 90% identical to AAGATGA (SEQ ID NO: 29). In some embodiments, theKozak sequence comprises AAGATGA (SEQ ID NO: 29). In some embodimentsthe Kozak sequence comprises a nucleic acid sequence at least 85%identical to GCAAGATG (SEQ ID NO: 44). In some embodiments the Kozaksequence differs from the sequence of GCAAGATG (SEQ ID NO: 44) by one ortwo nucleotides. In some embodiments, the Kozak sequence comprisesGCAAGATG (SEQ ID NO: 44). In some embodiments the Kozak sequencecomprises a nucleic acid sequence at least 85% identical to CACCATG (SEQID NO: 47). In some embodiments the Kozak sequence differs from thesequence of CACCATG (SEQ ID NO: 47) by one or two nucleotides. In someembodiments, the Kozak sequence comprises CACCATG (SEQ ID NO: 47). Insome embodiments, the nucleic acid construct further comprises a signalnucleic acid sequence encoding a signal peptide capable of increasingsecretion of the therapeutic protein as compared to the therapeuticprotein without the signal peptide. In some embodiments, the signalpeptide is selected from a binding immunoglobulin protein (BiP) signalpeptide and a Gaussia signal peptide. In some embodiments, the BiPsignal peptide comprises an amino acid sequence at least 90% identicalto an amino acid sequence selected from the group consisting of SEQ IDNOs: 13-17. In some embodiments, the BiP signal peptide comprises anamino acid sequence selected from the group consisting of SEQ ID NOs:13-17. In some embodiments, the Gaussia signal peptide comprises anamino acid sequence at least 90% identical to SEQ ID NO: 32. In someembodiments, the Gaussia signal peptide comprises SEQ ID NO: 32.

In some embodiments, any of the nucleic acid constructs provided hereinfurther comprise a nucleic acid sequence encoding a peptide thatselectively binds to the CI-MPR with high affinity, wherein thetherapeutic protein and the peptide that selectively binds to the CI-MPRare expressed as a fusion protein. In some embodiments, the nucleic acidconstruct further comprises a sequence encoding a linker peptide betweenthe nucleic acid encoding the peptide that selectively binds to theCI-MPR nucleotide sequence and the nucleic acid sequence encoding thetherapeutic protein. In some embodiments, the sequence of the linkerpeptide may overlap with the sequence of the therapeutic peptide or thesequence of the peptide that selectively binds to the CI-MPR, or both.In some embodiments, the peptide that binds to CI-MPR with high affinityis a variant IGF2 peptide (vIGF2). In some embodiments, the vIGF2peptide facilitates uptake into the cells. In some embodiments, thevIGF2 peptide comprises an amino acid sequence at least 90% identical toan amino acid sequence selected from the group consisting of SEQ ID NOs:1-11. In some embodiments, the vIGF2 peptide comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 2-11. In someembodiments, the vIGF2 nucleotide sequence is 5′ to the nucleic acidsequence encoding a therapeutic protein. In some embodiments, the vIGF2nucleotide sequence is 3′ to the nucleic acid sequence encoding atherapeutic protein. In some embodiments, the nucleic acid constructfurther comprises a sequence encoding a linker peptide between the vIGF2nucleotide sequence and the nucleic acid sequence encoding a therapeuticprotein. In some embodiments, the linker peptide consists of 5-20 aminoacids, 5-15 amino acids, 5-10 amino acids, 8-12 amino acids, or about 7,8, 9, 10, 11, 12 or 13 amino acids. In some embodiments, the linkerpeptide comprises an amino acid sequence at least 90% identical to SEQID NO: 18-21, SEQ ID NO: 33 or SEQ ID NO: 37. In some embodiments, thelinker peptide comprises SEQ ID NO: 18-21, SEQ ID NO: 33 or SEQ ID NO:37. In some embodiments, the therapeutic protein is associated with alysosomal storage disorder. In some embodiments, the therapeutic proteinis a lysosomal enzyme or enzymatically active fragment thereof. In someembodiments, the therapeutic protein is selected from the groupconsisting of alpha-galactosidase (A or B), β-galactosidase,β-hexosaminidase (A or B), galactosylceramidase, arylsulfatase (A or B),β-glucocerebrosidase, glucocerebrosidase, lysosomal acid lipase,lysosomal enzyme acid sphingomyelinase, formylglycine-generating enzyme,iduronidase (e.g., alpha-L), acetyl-CoA:alpha-glucosaminideN-acetyltransferase, glycosaminoglycan alpha-L-iduronohydrolase, heparanN-sulfatase, N-acetyl-α-D-glucosaminidase (NAGLU),iduronate-2-sulfatase, galactosamine-6-sulfate sulfatase,N-acetylgalactosamine-6-sulfatase, glycosaminoglycanN-acetylgalactosamine 4-sulfatase, (3-glucuronidase, hyaluronidase,alpha-N-acetyl neuraminidase (sialidase), ganglioside sialidase,phosphotransferase, alpha-glucosidase, alpha-D-mannosidase,beta-D-mannosidase, aspartylglucosaminidase, alpha-L-fucosidase,battenin, palmitoyl protein thioesterases, and other Batten-relatedproteins (e.g., ceroid-lipofuscinosis neuronal protein 6), or anenzymatically active fragment thereof. In some embodiments, thetherapeutic protein is an alpha-galactosidase, or an enzymaticallyactive fragment thereof. In some embodiments, the therapeutic protein isan alpha-glucosidase, or an enzymatically active fragment thereof. Insome embodiments, the therapeutic protein is a palmitoyl proteinthioesterase (PPT)—including palmitoyl protein thioesterase 1 and 2(PPT1 and PPT2 respectively). In some embodiments, the therapeuticprotein is palmitoyl protein thioesterase 1. In some embodiments, thetherapeutic protein is N-acetyl-α-D-glucosaminidase (NAGLU). In someembodiments, the therapeutic protein is associated with a geneticdisorder selected from the group consisting of CDKL5 deficiencydisorder, cystic fibrosis, alpha- and beta-thalassemias, sickle cellanemia, Marfan syndrome, fragile X syndrome, Huntington's disease,hemochromatosis, Congenital Deafness (nonsyndromic), Tay-Sachs, Familialhypercholesterolemia, Duchenne muscular dystrophy, Stargardt disease,Usher syndrome, choroideremia, achromatopsia, X-linked retinoschisis,hemophilia, Wiskott-Aldrich syndrome, X-linked chronic granulomatousdisease, aromatic L-amino acid decarboxylase deficiency, recessivedystrophic epidermolysis bullosa, alpha 1 antitrypsin deficiency,Hutchinson-Gilford progeria syndrome (HGPS), Noonan syndrome, X-linkedsevere combined immunodeficiency (X-SCID). In some embodiments, thetherapeutic protein is selected from the group consisting of CDKL5,Connexin 26, hexosaminidase A, LDL receptor, Dystrophin, CFTR,beta-globulin, HFE, Huntington, ABCA4, myosin VIIA (MYO7A), Rab escortprotein-1 (REP1), cyclic nucleotide gated channel beta 3 (CNGB3),retinoschisin 1 (RS1), hemoglobin subunit beta (HBB), Factor IX, WAS,cytochrome B-245 beta chain, dopa decarboxylase (DDC), collagen type VIIalpha 1 chain (COL7A1), serpin family A member 1 (SERPINA1), LMNA,PTPN11, SOS1, RAF1, KRAS, and IL2 receptor y gene. In some embodiments,the therapeutic protein is capable of replacing a defective or deficientprotein associated with a genetic disorder in a subject having thegenetic disorder. In some embodiments, the genetic disorder is alysosomal storage disorder. In some embodiments, the genetic disorder isselected from the group consisting of aspartylglucosaminuria, Battendisease, cystinosis, Fabry disease, Gaucher disease type I, Gaucherdisease type II, Gaucher disease type III, Pompe disease, Tay Sachsdisease, Sandhoff disease, metachomatic leukodystrophy, mucolipidosistype I, mucolipidosis type II, mucolipidosis type III, mucolipidosistype IV, Hurler disease, Hunter disease, Sanfilippo disease type A,Sanfilippo disease type B, Sanfilippo disease type C, Sanfilippo diseasetype D, Morquio disease type A, Morquio disease type B, Maroteau-Lamydisease, Sly disease, Niemann-Pick disease type A, Niemann-Pick diseasetype B, Niemann-Pick disease type C1, Niemann-Pick disease type C2,Schindler disease type I, and Schindler disease type II. In someembodiments, the lysosomal storage disorder is selected from the groupconsisting of activator deficiency, GM2-gangliosidosis;GM2-gangliosidosis, AB variant; alpha-mannosidosis (type 2, moderateform; type 3, neonatal, severe); beta-mannosidosis; lysosomal acidlipase deficiency; cystinosis (late-onset juvenile or adolescentnephropathic type; infantile nephropathic); Chanarin-Dorfman syndrome;neutral lipid storage disease with myopathy; NLSDM; Danon disease; Fabrydisease; Fabry disease type II, late-onset; Farber disease; Farberlipogranulomatosis; fucosidosis; galactosialidosis (combinedneuraminidase & beta-galactosidase deficiency); Gaucher disease; type IIGaucher disease; type III Gaucher disease; type IIIC Gaucher disease;Gaucher disease, atypical, due to saposin C deficiency;GM1-gangliosidosis (late-infantile/juvenile GM1-gangliosidosis;adult/chronic GM1-gangliosidosis); Globoid cell leukodystrophy, Krabbedisease (Late infantile onset; Juvenile Onset; Adult Onset); Krabbedisease, atypical, due to saposin A deficiency; MetachromaticLeukodystrophy (juvenile; adult); partial cerebroside sulfatedeficiency; pseudoarylsulfatase A deficiency; metachromaticleukodystrophy due to saposin B deficiency; Mucopolysaccharidosesdisorders: MPS I, Hurler syndrome; MPS I, Hurler-Scheie syndrome; MPS I,Scheie syndrome; MPS II, Hunter syndrome; MPS II, Hunter syndrome;Sanfilippo syndrome Type A/MPS IIIA; Sanfilippo syndrome Type B/MPSIIIB; Sanfilippo syndrome Type C/MPS IIIC; Sanfilippo syndrome TypeD/MPS IIID; Morquio syndrome, type A/MPS IVA; Morquio syndrome, typeB/MPS IVB; MPS IX hyaluronidase deficiency; MPS VI Maroteaux-Lamysyndrome; MPS VII Sly syndrome; mucolipidosis I, sialidosis type II;I-cell disease, Leroy disease, mucolipidosis II; Pseudo-Hurlerpolydystrophy/mucolipidosis type III; mucolipidosis IIIC/ML III GAMMA;mucolipidosis type IV; multiple sulfatase deficiency; Niemann-Pickdisease (type B; type C1/chronic neuronopathic form; type C2; typeD/Nova Scotian type); Neuronal Ceroid Lipofuscinoses: CLN6disease—Atypical Late Infantile, Late-Onset variant, Early Juvenile;Batten-Spielmeyer-Vogt/Juvenile NCL/CLN3 disease; Finnish Variant LateInfantile CLN5; Jansky-Bielschowsky disease/Late infantile CLN2/TPP1Disease; Kufs/Adult-onset NCL/CLN4 disease (type B); NorthernEpilepsy/variant late infantile CLN8; Santavuori-Haltia/InfantileCLN1/PPT disease; Pompe disease (glycogen storage disease type II);late-onset Pompe disease; Pycnodysostosis; Sandhoff disease/GM2gangliosidosis; Sandhoff disease/GM2 gangliosidosis; Sandhoffdisease/GM2 Gangliosidosis; Schindler disease (type III/intermediate,variable); Kanzaki disease; Salla disease; infantile free sialic acidstorage disease (ISSD); spinal muscular atrophy with progressivemyoclonic epilepsy (SMAPME); Tay-Sachs disease/GM2 gangliosidosis;juvenile-onset Tay-Sachs disease; late-onset Tay-Sachs disease;Christianson syndrome; Lowe oculocerebrorenal syndrome;Charcot-Marie-Tooth type 4J, CMT4J; Yunis-Varon syndrome; bilateraltemporooccipital polymicrogyria (BTOP); X-linked hypercalciuricnephrolithiasis, Dent-1; and Dent disease 2, adenosine deaminase severecombined immunodeficiency (ADA-SCID), and neuronal ceroidlipofuscinosis. In some embodiments, the genetic disorder is Pompedisease. In some embodiments, the genetic disorder is neuronal ceroidlipofuscinosis. In some embodiments, the neuronal ceroid lipofuscinosisis selected from the group consisting of Infantile NCL(Santavuori-Haltia disease), Late Infantile NCL (Jansky-Bielschowskydisease), Batten disease, Adult NCL (Kufs disease), Finnish LateInfantile NCL, Variant Late Infantile NCL, CLN7, CLN8, Turkish LateInfantile NCL, NCL type 9, and CLN10. In some embodiments, the genetherapy vector is a viral vector. In some embodiments, the viral vectoris an adeno-associated virus vector, a retrovirus vector, a lentivirusvector, a pox virus vector, a vaccinia virus vector, an adenovirusvector, or a herpes virus vector. In some embodiments, the viral vectoris an AAV vector. In some embodiments, the AAV vector comprises invertedterminal repeats (ITRs). In some embodiments, the AAV vector is selectedfrom the group consisting of an AAV1 vector, an AAV2 vector, an AAV3vector, an AAV4 vector, an AAV5 vector, an AAV6 vector, an AAV7 vector,an AAV8 vector, an AAV9 vector, an AAVrhS vector, an AAVrh10 vector, anAAVrh33 vector, an AAVrh34 vector, an AAVrh74 vector, an AAV Anc80vector, an AAVPHP.B vector, an AAVhu68 vector, and an AAV-DJ vector.

In certain aspects, there are provided gene therapy vectors, such asgene therapy vectors comprising: (a) a nucleic acid sequence encoding atherapeutic protein, and (b) a nucleic acid sequence encoding a peptidethat binds to the CI-MPR with high affinity. In some embodiments, thepeptide is a variant IGF2 (vIGF2) peptide. In some embodiments, thevIGF2 peptide comprises an amino acid sequence that is at least 90%identical to SEQ ID NO: 1 and having at least one substitution at one ormore positions selected from the group consisting of positions 6, 26,27, 43, 48, 49, 50, 54, 55, and 65 of SEQ ID NO: 1. In some embodiments,the at least one substitution is selected from the group consisting ofE6R, F26S, Y27L, V43L, F48T, R495, S50I, A54R, L55R, and K65R of SEQ IDNO: 1. In some embodiments, the vIGF2 peptide comprises at least twosubstitutions at two or more positions selected from the groupconsisting of positions 6, 26, 27, 43, 48, 49, 50, 54, and 55 of SEQ IDNO: 1. In some embodiments, the at least two substitutions are selectedfrom the group consisting of E6R, F26S, Y27L, V43L, F48T, R495, S50I,A54R, and L55R of SEQ ID NO: 1. In some embodiments, the vIGF2 peptidecomprises an N-terminal deletion. In some embodiments, the vIGF2 peptidecomprises an N-terminal deletion at position 1 of SEQ ID NO: 1. In someembodiments, the vIGF2 peptide comprises an N-terminal deletion ofpositions 1-2 of SEQ ID NO: 1. In some embodiments, the vIGF2 peptidecomprises an N-terminal deletion of positions 1-3 of SEQ ID NO: 1. Insome embodiments, the vIGF2 peptide comprises an N-terminal deletion ofpositions 1-4 of SEQ ID NO: 1. In some embodiments, the vIGF2 peptidecomprises an N-terminal deletion of positions 1-4 of SEQ ID NO: 1 andsubstitutions of E6R, Y27L, and K65R. In some embodiments, the vIGF2peptide comprises an N-terminal deletion of positions 1-4 of SEQ ID NO:1and substitutions of E6R and Y27L. In some embodiments, the vIGF2peptide comprises an N-terminal deletion of positions 1-5 of SEQ IDNO: 1. In some embodiments, the vIGF2 peptide comprises an N-terminaldeletion at positions 1-6 of SEQ ID NO: 1. In some embodiments, thevIGF2 peptide comprises an N-terminal deletion at positions 1-7 of SEQID NO: 1. In some embodiments, the vIGF2 peptide has decreased or noaffinity for the insulin receptor and IGF1R as compared to native IGF2peptide. In some embodiments, the vIGF2 peptide is capable offacilitating uptake of the therapeutic protein into a cell. In someembodiments, the vIGF2 peptide is capable of facilitating uptake of thetherapeutic protein into a lysosome. In some embodiments, thetherapeutic protein is capable of replacing a defective or deficientprotein associated with a genetic disorder in a subject having thegenetic disorder. In some embodiments, the therapeutic protein is alysosomal enzyme or enzymatically active fragment thereof. In someembodiments, the genetic disorder is a lysosomal storage disorder. Insome embodiments, the genetic disorder is selected from the groupconsisting of aspartylglucosaminuria, Batten disease, cystinosis, Fabrydisease, Gaucher disease type I, Gaucher disease type II, Gaucherdisease type III, Pompe disease, Tay Sachs disease, Sandhoff disease,metachomatic leukodystrophy, mucolipidosis type I, mucolipidosis typeII, mucolipidosis type III, mucolipidosis type IV, Hurler disease,Hunter disease, Sanfilippo disease type A, Sanfilippo disease type B,Sanfilippo disease type C, Sanfilippo disease type D, Morquio diseasetype A, Morquio disease type B, Maroteau-Lamy disease, Sly disease,Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pickdisease type C1, Niemann-Pick disease type C2, Schindler disease type I,and Schindler disease type II. In some embodiments, the lysosomalstorage disorder is selected from the group consisting of activatordeficiency, GM2-gangliosidosis; GM2-gangliosidosis, AB variant;alpha-mannosidosis (type 2, moderate form; type 3, neonatal, severe);beta-mannosidosis; lysosomal acid lipase deficiency; cystinosis(late-onset juvenile or adolescent nephropathic type; infantilenephropathic); Chanarin-Dorfman syndrome; neutral lipid storage diseasewith myopathy; NLSDM; Danon disease; Fabry disease; Fabry disease typeII, late-onset; Farber disease; Farber lipogranulomatosis; fucosidosis;galactosialidosis (combined neuraminidase & beta-galactosidasedeficiency); Gaucher disease; type II Gaucher disease; type III Gaucherdisease; type IIIC Gaucher disease; Gaucher disease, atypical, due tosaposin C deficiency; GM1-gangliosidosis (late-infantile/juvenileGM1-gangliosidosis; adult/chronic GM1-gangliosidosis); Globoid cellleukodystrophy, Krabbe disease (Late infantile onset; Juvenile Onset;Adult Onset); Krabbe disease, atypical, due to saposin A deficiency;Metachromatic Leukodystrophy (juvenile; adult); partial cerebrosidesulfate deficiency; pseudoarylsulfatase A deficiency; metachromaticleukodystrophy due to saposin B deficiency; Mucopolysaccharidosesdisorders: MPS I, Hurler syndrome; MPS I, Hurler-Scheie syndrome; MPS I,Scheie syndrome; MPS II, Hunter syndrome; MPS II, Hunter syndrome;Sanfilippo syndrome Type A/MPS IIIA; Sanfilippo syndrome Type B/MPSIIIB; Sanfilippo syndrome Type C/MPS IIIC; Sanfilippo syndrome TypeD/MPS IIID; Morquio syndrome, type A/MPS IVA; Morquio syndrome, typeB/MPS IVB; MPS IX hyaluronidase deficiency; MPS VI Maroteaux-Lamysyndrome; MPS VII Sly syndrome; mucolipidosis I, sialidosis type II;I-cell disease, Leroy disease, mucolipidosis II; Pseudo-Hurlerpolydystrophy/mucolipidosis type III; mucolipidosis IIIC/ML III GAMMA;mucolipidosis type IV; multiple sulfatase deficiency; Niemann-Pickdisease (type B; type C1/chronic neuronopathic form; type C2; typeD/Nova Scotian type); Neuronal Ceroid Lipofuscinoses: CLN6disease—Atypical Late Infantile, Late-Onset variant, Early Juvenile;Batten-Spielmeyer-Vogt/Juvenile NCL/CLN3 disease; Finnish Variant LateInfantile CLN5; Jansky-Bielschowsky disease/Late infantile CLN2/TPP1Disease; Kufs/Adult-onset NCL/CLN4 disease (type B); NorthernEpilepsy/variant late infantile CLN8; Santavuori-Haltia/InfantileCLN1/PPT disease; Pompe disease (glycogen storage disease type II);late-onset Pompe disease; Pycnodysostosis; Sandhoff disease/GM2gangliosidosis; Sandhoff disease/GM2 gangliosidosis; Sandhoffdisease/GM2 Gangliosidosis; Schindler disease (type III/intermediate,variable); Kanzaki disease; Salla disease; infantile free sialic acidstorage disease (ISSD); spinal muscular atrophy with progressivemyoclonic epilepsy (SMAPME); Tay-Sachs disease/GM2 gangliosidosis;juvenile-onset Tay-Sachs disease; late-onset Tay-Sachs disease;Christianson syndrome; Lowe oculocerebrorenal syndrome;Charcot-Marie-Tooth type 4J, CMT4J; Yunis-Varon syndrome; bilateraltemporooccipital polymicrogyria (BTOP); X-linked hypercalciuricnephrolithiasis, Dent-1; and Dent disease 2, adenosine deaminase severecombined immunodeficiency (ADA-SCID), chronic granulomatous disease(CGD), and neuronal ceroid lipofuscinosis. In some embodiments, thegenetic disorder is Pompe disease. In some embodiments, the geneticdisorder is neuronal ceroid lipofuscinosis. In some embodiments, theneuronal ceroid lipofuscinosis is selected from the group consisting ofInfantile NCL (Santavuori-Haltia disease), Late Infantile NCL(Jansky-Bielschowsky disease), Batten disease, Adult NCL (Kufs disease),Finnish Late Infantile NCL, Variant Late Infantile NCL, CLN7, CLN8,Turkish Late Infantile NCL, NCL type 9, and CLN10. In some embodiments,the therapeutic protein is a soluble lysosomal enzyme. In someembodiments, the therapeutic protein comprises an enzyme selected fromthe group consisting of alpha-galactosidase (A or B), β-galactosidase,β-hexosaminidase (A or B), galactosylceramidase, arylsulfatase (A or B),β-glucocerebrosidase, glucocerebrosidase, lysosomal acid lipase,lysosomal enzyme acid sphingomyelinase, formylglycine-generating enzyme,iduronidase (e.g., alpha-L), acetyl-CoA:alpha-glucosaminideN-acetyltransferase, glycosaminoglycan alpha-L-iduronohydrolase, heparanN-sulfatase, N-acetyl-α-D-glucosaminidase (NAGLU),iduronate-2-sulfatase, galactosamine-6-sulfate sulfatase,N-acetylgalactosamine-6-sulfatase, glycosaminoglycanN-acetylgalactosamine 4-sulfatase, β-glucuronidase, hyaluronidase,alpha-N-acetyl neuraminidase (sialidase), ganglioside sialidase,phosphotransferase, alpha-glucosidase, alpha-D-mannosidase,beta-D-mannosidase, aspartylglucosaminidase, alpha-L-fucosidase,battenin, palmitoyl protein thioesterases, and other Batten-relatedproteins (e.g., ceroid-lipofuscinosis neuronal protein 6), or anenzymatically active fragment thereof. In some embodiments, thetherapeutic protein is an alpha-glucosidase, or an enzymatically activefragment thereof. In some embodiments, the therapeutic protein isN-acetyl-α-D-glucosaminidase (NAGLU). In some embodiments, the palmitoylprotein thioesterase is palmitoyl protein thioesterase 1 (PPT1) or 2(PPT2). In some embodiments, the palmitoyl protein thioesterase ispalmitoyl protein thioesterase 1. In some embodiments, the nucleic acidconstruct further comprises a translation initiation sequence. In someembodiments, the translation initiation sequence comprises a Kozaksequence. In some embodiments, the Kozak sequence comprises the sequenceAX₁X₂ATGA (SEQ ID NO: 28), wherein each of X₁ and X₂ is any nucleotide.In some embodiments, X₁ comprises A. In some embodiments, X₂ comprisesG. In some embodiments, the Kozak sequence comprises a nucleic acidsequence at least 90% identical to AAGATGA (SEQ ID NO: 29). In someembodiments, the Kozak sequence comprises AAGATGA (SEQ ID NO: 29). Insome embodiments the Kozak sequence comprises a nucleic acid sequence atleast 85% identical to GCAAGATG (SEQ ID NO: 44). In some embodiments theKozak sequence differs from the sequence of GCAAGATG (SEQ ID NO: 44) byone or two nucleotides. In some embodiments, the Kozak sequencecomprises GCAAGATG (SEQ ID NO: 44). In some embodiments the Kozaksequence comprises a nucleic acid sequence at least 85% identical toCACCATG (SEQ ID NO: 47). In some embodiments the Kozak sequence differsfrom the sequence of CACCATG (SEQ ID NO: 47) by one or two nucleotides.In some embodiments, the Kozak sequence comprises CACCATG (SEQ ID NO:47). In some embodiments, the nucleic acid construct further comprises anucleic acid sequence encoding a signal peptide wherein the signalpeptide is capable of increasing secretion of the therapeutic protein ascompared to the therapeutic protein without the signal peptide. In someembodiments, the nucleic acid construct further comprises a nucleic acidsequence encoding a signal peptide wherein the signal peptide is capableof increasing secretion of the therapeutic protein as compared to thetherapeutic protein with the natural signal peptide. In someembodiments, the nucleic acid construct comprises a nucleic acidsequence encoding a non-native signal peptide, wherein the non-nativesignal peptide is capable of increasing secretion of the therapeuticprotein as compared to the native signal peptide for the therapeuticprotein. In some embodiments, the signal peptide is selected from abinding immunoglobulin protein (BiP) signal peptide and a Gaussia signalpeptide. In some embodiments, the BiP signal peptide comprises an aminoacid sequence at least 90% identical to an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 13-17. In some embodiments, theBiP signal peptide comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 13-17. In some embodiments, the signalpeptide comprises a Gaussia signal peptide. In some embodiments, theGaussia signal peptide comprises an amino acid sequence at least 90%identical to SEQ ID NO: 32. In some embodiments, the Gaussia signalpeptide comprises SEQ ID NO: 32. In some embodiments, the vIGF2 nucleicacid sequence is 5′ to the nucleic acid sequence encoding a therapeuticprotein. In some embodiments, the vIGF2 nucleic acid sequence is 3′ tothe nucleic acid sequence encoding a therapeutic protein. In someembodiments, the nucleic acid construct further comprises a sequenceencoding a linker peptide between the vIGF2 nucleotide sequence and thenucleic acid sequence encoding a therapeutic protein. In someembodiments, the linker consists of 5-20 amino acids, 5-15 amino acids,5-10 amino acids, 8-12 amino acids, or about 7, 8, 9, 10, 11, 12 or 13amino acids. In some embodiments, the linker peptide comprises SEQ IDNO: 18-21 or SEQ ID NO: 33. In some embodiments, the gene therapy vectoris a virus vector. In some embodiments, the virus vector is anadenovirus vector, an adeno-associated virus (AAV) vector, a retrovirusvector, a lentivirus vector, or a herpes virus vector. In someembodiments, the virus vector is an AAV vector. In some embodiments, theAAV vector comprises inverted terminal repeats (ITRs). In someembodiments, the AAV vector is selected from the group consisting of anAAV1 vector, an AAV2 vector, an AAV3 vector, an AAV4 vector, an AAV5vector, an AAV6 vector, an AAV7 vector, an AAV8 vector, an AAV9 vector,an AAVrhS vector, an AAVrh10 vector, an AAVrh33 vector, an AAVrh34vector, an AAVrh74 vector, an AAV Anc80 vector, an AAVPHP.B vector, anAAVhu68 vector, and an AAV-DJ vector.

A gene therapy vector comprising a nucleic acid construct comprising:(a) a nucleic acid sequence encoding a therapeutic protein, and (b) anucleic acid sequence encoding a peptide that increases endocytosis ofthe therapeutic protein. In some embodiments, the peptide that increasesendocytosis of the therapeutic protein is a peptide that binds to theCI-MPR. In some embodiments, the peptide is a variant IGF2 (vIGF2)peptide, a HIRMab, or a TfRMab or other cell targeting peptide orprotein. In some embodiments, the peptide is vIGF2. In some embodiments,the vIGF2 peptide comprises an amino acid sequence that is at least 90%identical to SEQ ID NO: 1 and having at least one substitution at one ormore positions selected from the group consisting of positions 6, 26,27, 43, 48, 49, 50, 54, 55, and 65 of SEQ ID NO: 1. In some embodiments,the at least one substitution is selected from the group consisting ofE6R, F26S, Y27L, V43L, F48T, R495, S50I, A54R, L55R, and K65R of SEQ IDNO: 1. In some embodiments, the at least one substitution is selectedfrom the group consisting of E6R, F26S, Y27L, V43L, F48T, R495, S50I,A54R, and L55R of SEQ ID NO: 1. In some embodiments, the vIGF2 peptidecomprises at least two substitutions at two or more positions selectedfrom the group consisting of positions 6, 26, 27, 43, 48, 49, 50, 54,and 55 of SEQ ID NO: 1. In some embodiments, the at least twosubstitutions are selected from the group consisting of E6R, F26S, Y27L,V43L, F48T, R495, S50I, A54R, and L55R of SEQ ID NO: 1. In someembodiments, the vIGF2 peptide comprises an N-terminal deletion. In someembodiments, the vIGF2 peptide comprises an N-terminal deletion atposition 1 of SEQ ID NO: 1. In some embodiments, the vIGF2 peptidecomprises an N-terminal deletion at positions 1-6 of SEQ ID NO: 1. Insome embodiments, the vIGF2 peptide comprises an N-terminal deletion ofpositions 1-2 of SEQ ID NO: 1. In some embodiments, the vIGF2 peptidecomprises an N-terminal deletion of positions 1-3 of SEQ ID NO: 1. Insome embodiments, the vIGF2 peptide comprises an N-terminal deletion ofpositions 1-4 of SEQ ID NO: 1. In some embodiments, the vIGF2 peptidecomprises an N-terminal deletion of positions 1-4 of SEQ ID NO: 1 and asubstitution of E6R, Y27L, and K65R. In some embodiments, the vIGF2peptide comprises an N-terminal deletion of positions 1-4 of SEQ ID NO:1and a substitution of E6R and Y27L. In some embodiments, the vIGF2peptide comprises an N-terminal deletion of positions 1-5 of SEQ IDNO: 1. In some embodiments, the vIGF2 peptide comprises an N-terminaldeletion of positions 1-6 of SEQ ID NO: 1. In some embodiments, thevIGF2 peptide comprises an N-terminal deletion at positions 1-7 of SEQID NO: 1. In some embodiments, the vIGF2 peptide has increasedspecificity for the cation-independent M6P receptor (CI-MPR) as comparedto native IGF2 peptide. In some embodiments, the vIGF2 peptide iscapable of facilitating uptake of the therapeutic protein into alysosome in a cell. In some embodiments, the therapeutic protein iscapable of replacing a defective or deficient protein associated with agenetic disorder in a subject having the genetic disorder. In someembodiments, the therapeutic protein is a lysosomal enzyme orenzymatically active fragment thereof. In some embodiments, the geneticdisorder is a lysosomal storage disorder. In some embodiments, thegenetic disorder is selected from the group consisting ofaspartylglucosaminuria, Batten disease, cystinosis, Fabry disease,Gaucher disease type I, Gaucher disease type II, Gaucher disease typeIII, Pompe disease, Tay Sachs disease, Sandhoff disease, metachomaticleukodystrophy, mucolipidosis type I, mucolipidosis type II,mucolipidosis type III, mucolipidosis type IV, Hurler disease, Hunterdisease, Sanfilippo disease type A, Sanfilippo disease type B,Sanfilippo disease type C, Sanfilippo disease type D, Morquio diseasetype A, Morquio disease type B, Maroteau-Lamy disease, Sly disease,Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pickdisease type C1, Niemann-Pick disease type C2, Schindler disease type I,and Schindler disease type II. In some embodiments, the lysosomalstorage disorder is selected from the group consisting of activatordeficiency, GM2-gangliosidosis; GM2-gangliosidosis, AB variant;alpha-mannosidosis (type 2, moderate form; type 3, neonatal, severe);beta-mannosidosis; lysosomal acid lipase deficiency; cystinosis(late-onset juvenile or adolescent nephropathic type; infantilenephropathic); Chanarin-Dorfman syndrome; neutral lipid storage diseasewith myopathy; NLSDM; Danon disease; Fabry disease; Fabry disease typeII, late-onset; Farber disease; Farber lipogranulomatosis; fucosidosis;galactosialidosis (combined neuraminidase & beta-galactosidasedeficiency); Gaucher disease; type II Gaucher disease; type III Gaucherdisease; type IIIC Gaucher disease; Gaucher disease, atypical, due tosaposin C deficiency; GM1-gangliosidosis (late-infantile/juvenileGM1-gangliosidosis; adult/chronic GM1-gangliosidosis); Globoid cellleukodystrophy, Krabbe disease (Late infantile onset; Juvenile Onset;Adult Onset); Krabbe disease, atypical, due to saposin A deficiency;Metachromatic Leukodystrophy (juvenile; adult); partial cerebrosidesulfate deficiency; pseudoarylsulfatase A deficiency; metachromaticleukodystrophy due to saposin B deficiency; Mucopolysaccharidosesdisorders: MPS I, Hurler syndrome; MPS I, Hurler-Scheie syndrome; MPS I,Scheie syndrome; MPS II, Hunter syndrome; MPS II, Hunter syndrome;Sanfilippo syndrome Type A/MPS IIIA; Sanfilippo syndrome Type B/MPSIIIB; Sanfilippo syndrome Type C/MPS IIIC; Sanfilippo syndrome TypeD/MPS IIID; Morquio syndrome, type A/MPS IVA; Morquio syndrome, typeB/MPS IVB; MPS IX hyaluronidase deficiency; MPS VI Maroteaux-Lamysyndrome; MPS VII Sly syndrome; mucolipidosis I, sialidosis type II;I-cell disease, Leroy disease, mucolipidosis II; Pseudo-Hurlerpolydystrophy/mucolipidosis type III; mucolipidosis IIIC/ML III GAMMA;mucolipidosis type IV; multiple sulfatase deficiency; Niemann-Pickdisease (type B; type C1/chronic neuronopathic form; type C2; typeD/Nova Scotian type); Neuronal Ceroid Lipofuscinoses: CLN6disease—Atypical Late Infantile, Late-Onset variant, Early Juvenile;Batten-Spielmeyer-Vogt/Juvenile NCL/CLN3 disease; Finnish Variant LateInfantile CLN5; Jansky-Bielschowsky disease/Late infantile CLN2/TPP1Disease; Kufs/Adult-onset NCL/CLN4 disease (type B); NorthernEpilepsy/variant late infantile CLN8; Santavuori-Haltia/InfantileCLN1/PPT disease; Pompe disease (glycogen storage disease type II);late-onset Pompe disease; Pycnodysostosis; Sandhoff disease/GM2gangliosidosis; Sandhoff disease/GM2 gangliosidosis; Sandhoffdisease/GM2 Gangliosidosis; Schindler disease (type III/intermediate,variable); Kanzaki disease; Salla disease; infantile free sialic acidstorage disease (ISSD); spinal muscular atrophy with progressivemyoclonic epilepsy (SMAPME); Tay-Sachs disease/GM2 gangliosidosis;juvenile-onset Tay-Sachs disease; late-onset Tay-Sachs disease;Christianson syndrome; Lowe oculocerebrorenal syndrome;Charcot-Marie-Tooth type 4J, CMT4J; Yunis-Varon syndrome; bilateraltemporooccipital polymicrogyria (BTOP); X-linked hypercalciuricnephrolithiasis, Dent-1; and Dent disease 2, adenosine deaminase severecombined immunodeficiency (ADA-SCID), chronic granulomatous disease(CGD), and neuronal ceroid lipofuscinosis. In some embodiments, thegenetic disorder is Pompe disease. In some embodiments, the geneticdisorder is neuronal ceroid lipofuscinosis. In some embodiments, theneuronal ceroid lipofuscinosis is selected from the group consisting ofInfantile NCL (Santavuori-Haltia disease), Late Infantile NCL(Jansky-Bielschowsky disease), Batten disease, Adult NCL (Kufs disease),Finnish Late Infantile NCL, Variant Late Infantile NCL, CLN7, CLN8,Turkish Late Infantile NCL, NCL type 9, and CLN10. In some embodiments,the therapeutic protein is a soluble lysosomal enzyme or anenzymatically active fragment thereof. In some embodiments, thetherapeutic protein comprises an enzyme selected from the groupconsisting of alpha-galactosidase (A or B), β-galactosidase,β-hexosaminidase (A or B), galactosylceramidase, arylsulfatase (A or B),β-glucocerebrosidase, glucocerebrosidase, lysosomal acid lipase,lysosomal enzyme acid sphingomyelinase, formylglycine-generating enzyme,iduronidase (e.g., alpha-L), acetyl-CoA:alpha-glucosaminideN-acetyltransferase, glycosaminoglycan alpha-L-iduronohydrolase, heparanN-sulfatase, N-acetyl-α-D-glucosaminidase (NAGLU),iduronate-2-sulfatase, galactosamine-6-sulfate sulfatase,N-acetylgalactosamine-6-sulfatase, glycosaminoglycanN-acetylgalactosamine 4-sulfatase, β-glucuronidase, hyaluronidase,alpha-N-acetyl neuraminidase (sialidase), ganglioside sialidase,phosphotransferase, alpha-glucosidase, alpha-D-mannosidase,beta-D-mannosidase, aspartylglucosaminidase, alpha-L-fucosidase,battenin, palmitoyl protein thioesterases, and other Batten-relatedproteins (e.g., ceroid-lipofuscinosis neuronal protein 6), or anenzymatically active fragment thereof. In some embodiments, thetherapeutic protein is an alpha-glucosidase, or an enzymatically activefragment thereof. In some embodiments, the therapeutic protein isN-acetyl-α-D-glucosaminidase (NAGLU). In some embodiments, the palmitoylprotein thioesterase is palmitoyl protein thioesterase 1 (PPT1) or 2(PPT2). In some embodiments, the palmitoyl protein thioesterase ispalmitoyl protein thioesterase 1. In some embodiments, the nucleic acidconstruct further comprises a translation initiation sequence. In someembodiments, the translation initiation sequence comprises a Kozaksequence. In some embodiments, the Kozak sequence comprises the sequenceAX₁X₂ATGA (SEQ ID NO: 28), wherein each of X₁ and X₂ is any nucleotide.In some embodiments, X₁ comprises A. In some embodiments, X₂ comprisesG. In some embodiments, the Kozak sequence comprises a nucleic acidsequence at least 90% identical to AAGATGA (SEQ ID NO: 29). In someembodiments, the Kozak sequence comprises AAGATGA (SEQ ID NO: 29). Insome embodiments the Kozak sequence comprises a nucleic acid sequence atleast 85% identical to GCAAGATG (SEQ ID NO: 44). In some embodiments theKozak sequence differs from the sequence of GCAAGATG (SEQ ID NO: 44) byone or two nucleotides. In some embodiments, the Kozak sequencecomprises GCAAGATG (SEQ ID NO: 44). In some embodiments the Kozaksequence comprises a nucleic acid sequence at least 85% identical toCACCATG (SEQ ID NO: 47). In some embodiments the Kozak sequence differsfrom the sequence of CACCATG (SEQ ID NO: 47) by one or two nucleotides.In some embodiments, the Kozak sequence comprises CACCATG (SEQ ID NO:47). In some embodiments, the nucleic acid construct further comprises asignal nucleic acid sequence encoding a signal peptide wherein thesignal peptide is capable of increasing secretion of the therapeuticprotein as compared to the therapeutic protein without the signalpeptide. In some embodiments, the signal peptide is selected from abinding immunoglobulin protein (BiP) signal peptide and a Gaussia signalpeptide. In some embodiments, the BiP signal peptide comprises an aminoacid sequence at least 90% identical to an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 13-17. In some embodiments, theBiP signal peptide comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 13-17. In some embodiments, the signalpeptide comprises a Gaussia signal peptide. In some embodiments, theGaussia signal peptide comprises an amino acid sequence at least 90%identical to SEQ ID NO: 32. In some embodiments, the Gaussia signalpeptide comprises SEQ ID NO: 32. In some embodiments, the vIGF2 nucleicacid sequence is 5′ to the nucleic acid sequence encoding a therapeuticprotein. In some embodiments, the vIGF2 nucleic acid sequence is 3′ tothe nucleic acid sequence encoding a therapeutic protein. In someembodiments, the nucleic acid construct further comprises a linkersequence encoding a linker peptide between the vIGF2 nucleotide sequenceand the nucleic acid sequence encoding a therapeutic protein. In someembodiments, the linker consists of 5-20 amino acids, 5-15 amino acids,5-10 amino acids, 8-12 amino acids, or about 7, 8, 9, 10, 11, 12 or 13amino acids. In some embodiments, the linker peptide comprises SEQ IDNO: 18-21 or SEQ ID NO: 33. In some embodiments, the gene therapy vectoris a virus vector. In some embodiments, the virus vector is anadenovirus vector, an adeno-associated virus (AAV) vector, a retrovirusvector, a lentivirus vector, or a herpes virus vector. In someembodiments, the virus vector is an AAV vector. In some embodiments, theAAV vector comprises inverted terminal repeats (ITRs). In someembodiments, the AAV vector is selected from the group consisting of anAAV1 vector, an AAV2 vector, an AAV3 vector, an AAV4 vector, an AAV5vector, an AAV6 vector, an AAV7 vector, an AAV8 vector, an AAV9 vector,an AAVrhS vector, an AAVrh10 vector, an AAVrh33 vector, an AAVrh34vector, an AAVrh74 vector, an AAV Anc80 vector, an AAVPHP.B vector, anAAVhu68 vector, and an AAV-DJ vector.

In additional aspects, there are provided pharmaceutical compositioncomprising (i) a therapeutically effective amount of any one of the genetherapy vectors herein and (ii) a pharmaceutically acceptable carrier orexcipient. In some embodiments, the carrier or excipient comprises anon-ionic, low-osmolar compound, a buffer, a polymer, a salt, or acombination thereof.

In further aspects, there are provided methods for treating a geneticdisorder comprising administering to a subject in need thereof anyone ofthe gene therapy vectors provided herein or the any one of thepharmaceutical compositions provided herein. In some embodiments, thegenetic disorder is a lysosomal storage disorder. In some embodiments,the genetic disorder is selected from the group consisting ofaspartylglucosaminuria, Batten disease, cystinosis, Fabry disease,Gaucher disease type I, Gaucher disease type II, Gaucher disease typeIII, Pompe disease, Tay Sachs disease, Sandhoff disease, metachomaticleukodystrophy, mucolipidosis type I, mucolipidosis type II,mucolipidosis type III, mucolipidosis type IV, Hurler disease, Hunterdisease, Sanfilippo disease type A, Sanfilippo disease type B,Sanfilippo disease type C, Sanfilippo disease type D, Morquio diseasetype A, Morquio disease type B, Maroteau-Lamy disease, Sly disease,Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pickdisease type C1, Niemann-Pick disease type C2, Schindler disease type I,and Schindler disease type II. In some embodiments, the lysosomalstorage disorder is selected from the group consisting of activatordeficiency, GM2-gangliosidosis; GM2-gangliosidosis, AB variant;alpha-mannosidosis (type 2, moderate form; type 3, neonatal, severe);beta-mannosidosis; lysosomal acid lipase deficiency; cystinosis(late-onset juvenile or adolescent nephropathic type; infantilenephropathic); Chanarin-Dorfman syndrome; neutral lipid storage diseasewith myopathy; NLSDM; Danon disease; Fabry disease; Fabry disease typeII, late-onset; Farber disease; Farber lipogranulomatosis; fucosidosis;galactosialidosis (combined neuraminidase & beta-galactosidasedeficiency); Gaucher disease; type II Gaucher disease; type III Gaucherdisease; type IIIC Gaucher disease; Gaucher disease, atypical, due tosaposin C deficiency; GM1-gangliosidosis (late-infantile/juvenileGM1-gangliosidosis; adult/chronic GM1-gangliosidosis); Globoid cellleukodystrophy, Krabbe disease (Late infantile onset; Juvenile Onset;Adult Onset); Krabbe disease, atypical, due to saposin A deficiency;Metachromatic Leukodystrophy (juvenile; adult); partial cerebrosidesulfate deficiency; pseudoarylsulfatase A deficiency; metachromaticleukodystrophy due to saposin B deficiency; Mucopolysaccharidosesdisorders: MPS I, Hurler syndrome; MPS I, Hurler-Scheie syndrome; MPS I,Scheie syndrome; MPS II, Hunter syndrome; MPS II, Hunter syndrome;Sanfilippo syndrome Type A/MPS IIIA; Sanfilippo syndrome Type B/MPSIIIB; Sanfilippo syndrome Type C/MPS IIIC; Sanfilippo syndrome TypeD/MPS IIID; Morquio syndrome, type A/MPS IVA; Morquio syndrome, typeB/MPS IVB; MPS IX hyaluronidase deficiency; MPS VI Maroteaux-Lamysyndrome; MPS VII Sly syndrome; mucolipidosis I, sialidosis type II;I-cell disease, Leroy disease, mucolipidosis II; Pseudo-Hurlerpolydystrophy/mucolipidosis type III; mucolipidosis IIIC/ML III GAMMA;mucolipidosis type IV; multiple sulfatase deficiency; Niemann-Pickdisease (type B; type C1/chronic neuronopathic form; type C2; typeD/Nova Scotian type); Neuronal Ceroid Lipofuscinoses: CLN6disease—Atypical Late Infantile, Late-Onset variant, Early Juvenile;Batten-Spielmeyer-Vogt/Juvenile NCL/CLN3 disease; Finnish Variant LateInfantile CLN5; Jansky-Bielschowsky disease/Late infantile CLN2/TPP1Disease; Kufs/Adult-onset NCL/CLN4 disease (type B); NorthernEpilepsy/variant late infantile CLN8; Santavuori-Haltia/InfantileCLN1/PPT disease; Pompe disease (glycogen storage disease type II);late-onset Pompe disease; Pycnodysostosis; Sandhoff disease/GM2gangliosidosis; Sandhoff disease/GM2 gangliosidosis; Sandhoffdisease/GM2 Gangliosidosis; Schindler disease (type III/intermediate,variable); Kanzaki disease; Salla disease; infantile free sialic acidstorage disease (ISSD); spinal muscular atrophy with progressivemyoclonic epilepsy (SMAPME); Tay-Sachs disease/GM2 gangliosidosis;juvenile-onset Tay-Sachs disease; late-onset Tay-Sachs disease;Christianson syndrome; Lowe oculocerebrorenal syndrome;Charcot-Marie-Tooth type 4J, CMT4J; Yunis-Varon syndrome; bilateraltemporooccipital polymicrogyria (BTOP); X-linked hypercalciuricnephrolithiasis, Dent-1; and Dent disease 2, adenosine deaminase severecombined immunodeficiency (ADA-SCID), chronic granulomatous disease(CGD), CDKL5 deficiency disorder, and neuronal ceroid lipofuscinosis. Insome embodiments, the genetic disorder is Pompe disease. In someembodiments, the genetic disorder is neuronal ceroid lipofuscinosis. Insome embodiments, the neuronal ceroid lipofuscinosis is selected fromthe group consisting of Infantile NCL (Santavuori-Haltia disease), LateInfantile NCL (Jansky-Bielschowsky disease), Batten disease, Adult NCL(Kufs disease), Finnish Late Infantile NCL, Variant Late Infantile NCL,CLN7, CLN8, Turkish Late Infantile NCL, NCL type 9, and CLN10. In someembodiments, the administering is performed intrathecally,intraocularly, intravitreally, retinally, intravenously,intramuscularly, intraventricularly, intracerebrally, intracerebellarly,intracerebroventricularly, intraperenchymally, ocularly, subcutaneously,or a combination thereof. In some embodiments, the administering isperformed intrathecally. In some embodiments, the administering isperformed intraocularly, intravitreally, or retinally.

In additional aspects, there are provided pharmaceutical compositionscomprising any one of the gene therapy vectors herein and apharmaceutically acceptable carrier or excipient for use in treating agenetic disorder. In further aspects, there are provided pharmaceuticalcompositions comprising any one of the gene therapy vectors herein and apharmaceutically acceptable carrier or excipient for use in preparationof a medicament for treatment of a genetic disorder. In someembodiments, the genetic disorder is a lysosomal storage disorder. Insome embodiments, the genetic disorder is selected from the groupconsisting of aspartylglucosaminuria, Batten disease, cystinosis, Fabrydisease, Gaucher disease type I, Gaucher disease type II, Gaucherdisease type III, Pompe disease, Tay Sachs disease, Sandhoff disease,metachomatic leukodystrophy, mucolipidosis type I, mucolipidosis typeII, mucolipidosis type III, mucolipidosis type IV, Hurler disease,Hunter disease, Sanfilippo disease type A, Sanfilippo disease type B,Sanfilippo disease type C, Sanfilippo disease type D, Morquio diseasetype A, Morquio disease type B, Maroteau-Lamy disease, Sly disease,Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pickdisease type C1, Niemann-Pick disease type C2, Schindler disease type I,and Schindler disease type II. In some embodiments, the lysosomalstorage disorder is selected from the group consisting of activatordeficiency, GM2-gangliosidosis; GM2-gangliosidosis, AB variant;alpha-mannosidosis (type 2, moderate form; type 3, neonatal, severe);beta-mannosidosis; lysosomal acid lipase deficiency; cystinosis(late-onset juvenile or adolescent nephropathic type; infantilenephropathic); Chanarin-Dorfman syndrome; neutral lipid storage diseasewith myopathy; NLSDM; Danon disease; Fabry disease; Fabry disease typeII, late-onset; Farber disease; Farber lipogranulomatosis; fucosidosis;galactosialidosis (combined neuraminidase & beta-galactosidasedeficiency); Gaucher disease; type II Gaucher disease; type III Gaucherdisease; type IIIC Gaucher disease; Gaucher disease, atypical, due tosaposin C deficiency; GM1-gangliosidosis (late-infantile/juvenileGM1-gangliosidosis; adult/chronic GM1-gangliosidosis); Globoid cellleukodystrophy, Krabbe disease (Late infantile onset; Juvenile Onset;Adult Onset); Krabbe disease, atypical, due to saposin A deficiency;Metachromatic Leukodystrophy (juvenile; adult); partial cerebrosidesulfate deficiency; pseudoarylsulfatase A deficiency; metachromaticleukodystrophy due to saposin B deficiency; Mucopolysaccharidosesdisorders: MPS I, Hurler syndrome; MPS I, Hurler-Scheie syndrome; MPS I,Scheie syndrome; MPS II, Hunter syndrome; MPS II, Hunter syndrome;Sanfilippo syndrome Type A/MPS IIIA; Sanfilippo syndrome Type B/MPSIIIB; Sanfilippo syndrome Type C/MPS IIIC; Sanfilippo syndrome TypeD/MPS IIID; Morquio syndrome, type A/MPS IVA; Morquio syndrome, typeB/MPS IVB; MPS IX hyaluronidase deficiency; MPS VI Maroteaux-Lamysyndrome; MPS VII Sly syndrome; mucolipidosis I, sialidosis type II;I-cell disease, Leroy disease, mucolipidosis II; Pseudo-Hurlerpolydystrophy/mucolipidosis type III; mucolipidosis IIIC/ML III GAMMA;mucolipidosis type IV; multiple sulfatase deficiency; Niemann-Pickdisease (type B; type C1/chronic neuronopathic form; type C2; typeD/Nova Scotian type); Neuronal Ceroid Lipofuscinoses: CLN6disease—Atypical Late Infantile, Late-Onset variant, Early Juvenile;Batten-Spielmeyer-Vogt/Juvenile NCL/CLN3 disease; Finnish Variant LateInfantile CLN5; Jansky-Bielschowsky disease/Late infantile CLN2/TPP1Disease; Kufs/Adult-onset NCL/CLN4 disease (type B); NorthernEpilepsy/variant late infantile CLN8; Santavuori-Haltia/InfantileCLN1/PPT disease; Pompe disease (glycogen storage disease type II);late-onset Pompe disease; Pycnodysostosis; Sandhoff disease/GM2gangliosidosis; Sandhoff disease/GM2 gangliosidosis; Sandhoffdisease/GM2 Gangliosidosis; Schindler disease (type III/intermediate,variable); Kanzaki disease; Salla disease; infantile free sialic acidstorage disease (ISSD); spinal muscular atrophy with progressivemyoclonic epilepsy (SMAPME); Tay-Sachs disease/GM2 gangliosidosis;juvenile-onset Tay-Sachs disease; late-onset Tay-Sachs disease;Christianson syndrome; Lowe oculocerebrorenal syndrome;Charcot-Marie-Tooth type 4J, CMT4J; Yunis-Varon syndrome; bilateraltemporooccipital polymicrogyria (BTOP); X-linked hypercalciuricnephrolithiasis, Dent-1; and Dent disease 2, adenosine deaminase severecombined immunodeficiency (ADA-SCID), chronic granulomatous disease(CGD), CDKL5 deficiency disorder, and neuronal ceroid lipofuscinosis. Insome embodiments, the genetic disorder is Pompe disease. In someembodiments, the genetic disorder is neuronal ceroid lipofuscinosis. Insome embodiments, the neuronal ceroid lipofuscinosis is selected fromthe group consisting of Infantile NCL (Santavuori-Haltia disease), LateInfantile NCL (Jansky-Bielschowsky disease), Batten disease, Adult NCL(Kufs disease), Finnish Late Infantile NCL, Variant Late Infantile NCL,CLN7, CLN8, Turkish Late Infantile NCL, NCL type 9, and CLN10. In someembodiments, the composition is formulated for administrationintrathecally, intraocularly, intravitreally, retinally, intravenously,intramuscularly, intraventricularly, intracerebrally, intracerebellarly,ocularly, or subcutaneously. In some embodiments, the composition isformulated for administration intrathecally. In some embodiments, thecomposition is formulated for administration intrathecally for treatinga neurodegenerative disorder. In some embodiments, the composition isformulated for administration ocularly, intravitreally, or retinally.

Provided herein are gene therapy vectors comprising a nucleic acidconstruct encoding a polypeptide comprising: (a) a therapeutic protein;(b) a peptide that binds to the cation-independent mannose 6-phosphate(M6P) receptor (CI-MPR) with high affinity; and (c) a linker between thetherapeutic protein and the peptide that binds CI-MPR. In someembodiments, the peptide is a variant IGF2 (vIGF2) peptide. In someembodiments, the vIGF2 peptide comprises an amino acid sequence that isat least 90% identical to SEQ ID NO: 1 and having at least onesubstitution at one or more positions selected from the group consistingof positions 6, 26, 27, 43, 48, 49, 50, 54, 55, and 65 of SEQ ID NO: 1.In some embodiments, the at least one substitution is selected from thegroup consisting of E6R, F26S, Y27L, V43L, F48T, R495, S50I, A54R, L55R,and K65R of SEQ ID NO:1. In some embodiments, the vIGF2 peptidecomprises at least two substitutions at two or more positions selectedfrom the group consisting of positions 6, 26, 27, 43, 48, 49, 50, 54,55, 65 of SEQ ID NO: 1. In some embodiments, the at least twosubstitutions are selected from the group consisting of E6R, F26S, Y27L,V43L, F48T, R495, S50I, A54R, L55R, K65R of SEQ ID NO: 1. In someembodiments, the vIGF2 peptide comprises an N-terminal deletion atpositions 1-4 of SEQ ID NO: 1. In some embodiments, wherein the vIGF2peptide has decreased affinity for insulin receptor and IGF1R ascompared to native IGF2 peptide. In some embodiments, the vIGF2 peptideis capable of facilitating uptake of the therapeutic protein into acell. In some embodiments, the vIGF2 peptide is capable of facilitatinguptake of the therapeutic protein into a lysosome. In some embodiments,the therapeutic protein is capable of replacing a defective or deficientprotein associated with a genetic disorder in a subject having thegenetic disorder. In some embodiments, genetic disorder is a lysosomalstorage disorder. In some embodiments, the genetic disorder is selectedfrom the group consisting of aspartylglucosaminuria, Batten disease,cystinosis, Fabry disease, Gaucher disease type I, Gaucher disease typeII, Gaucher disease type III, Pompe disease, Tay Sachs disease, Sandhoffdisease, metachomatic leukodystrophy, mucolipidosis type I,mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV,Hurler disease, Hunter disease, Sanfilippo disease type A, Sanfilippodisease type B, Sanfilippo disease type C, Sanfilippo disease type D,Morquio disease type A, Morquio disease type B, Maroteau-Lamy disease,Sly disease, Niemann-Pick disease type A, Niemann-Pick disease type B,Niemann-Pick disease type C1, Niemann-Pick disease type C2, Schindlerdisease type I, Schindler disease type II, adenosine deaminase severecombined immunodeficiency (ADA-SCID), chronic granulomatous disease(CGD), and neuronal ceroid lipofuscinosis. In some embodiments, thegenetic disorder is Pompe disease. In some embodiments, the geneticdisorder is a CLN1 disease. In some embodiments, the therapeutic proteincomprises a soluble lysosomal enzyme or an enzymatically active fragmentthereof. In some embodiments, the therapeutic protein comprises alysosomal enzyme or an enzymatically active fragment thereof, whereinthe lysosomal enzyme is selected from the group consisting ofalpha-galactosidase A, β-glucocerebrosidase, glucocerebrosidase,lysosomal acid lipase, glycosaminoglycan alpha-L-iduronohydrolase,iduronate-2-sulfatase, N-acetylgalactosamine-6-sulfatase,glycosaminoglycan N-acetylgalactosamine 4-sulfatase, palmitoyl proteinthioesterases, cyclin dependent kinase like 5, and alpha-glucosidase. Insome embodiments, the therapeutic protein is alpha-glucosidase or anenzymatically active fragment thereof. In some embodiments, thetherapeutic protein is a palmitoyl protein thioesterase or anenzymatically active fragment thereof. In some embodiments, thetherapeutic protein is palmitoyl protein thioesterase-1 or anenzymatically active fragment thereof. In some embodiments, the nucleicacid construct further comprises a translation initiation sequence. Insome embodiments, the translation initiation sequence comprises a Kozaksequence. In some embodiments, the nucleic acid construct furthercomprises a nucleic acid sequence encoding a signal peptide wherein thesignal peptide is capable of increasing secretion of the therapeuticprotein as compared to the therapeutic protein without the signalpeptide. In some embodiments, the signal peptide is selected from abinding immunoglobulin protein (BiP) signal peptide and a Gaussia signalpeptide. In some embodiments, the BiP signal peptide comprises an aminoacid sequence at least 90% identical to an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 13-17. In some embodiments, thevIGF2 peptide comprises the sequence of SEQ ID NO:31. In someembodiments, the construct comprises SEQ ID NO:36. In some embodiments,the polypeptide comprises SEQ ID NO:23. In some embodiments, theconstruct comprises SEQ ID NO:38. In some embodiments, the vIGF2 at theN-terminus of the polypeptide. In some embodiments, the vIGF2 is at theC-terminus of the polypeptide. In some embodiments, the linker peptidecomprises SEQ ID NO: 18-21 or SEQ ID NO: 33. In some embodiments, thegene therapy vector is a virus vector selected from the group consistingof an adenovirus vector, an adeno-associated virus (AAV) vector, aretrovirus vector, a lentivirus vector, a pox virus vector, a vacciniavirus vector, an adenovirus vector, and a herpes virus vector.

In certain aspects, there are provided fusion proteins, such as fusionproteins comprising a variant IGF2 peptide and a therapeutic protein. Insome embodiments, the fusion protein further comprises a linker. In someembodiments, the linker consists of 5-20 amino acids, 5-15 amino acids,5-10 amino acids, 8-12 amino acids, or about 7, 8, 9, 10, 11, 12 or 13amino acids. In some embodiments, the linker comprises an amino acidsequence of GGGGSGGGG (SEQ ID NO: 18), GGGGS (SEQ ID NO: 19), GGGSGGGGS(SEQ ID NO: 20), GGGGSGGGS (SEQ ID NO: 21), GGGGSGGGGS (SEQ ID NO: 37,also referred to herein as “2GS”), or GGSGSGSTS (SEQ ID NO: 33). In someembodiments, the fusion protein further comprises a signal peptide. Insome embodiments, the signal peptide comprises a binding immunoglobulinprotein (BiP) signal peptide. In some embodiments, the fusion protein isencoded by a nucleic acid comprising a cricket paralysis virus internalribosome entry sequence (CrPV IBES). In some embodiments, thetherapeutic protein comprises at least one enzyme of the groupconsisting of alpha-galactosidase (A or B), β-galactosidase,β-hexosaminidase (A or B), galactosylceramidase, arylsulfatase (A or B),β-glucocerebrosidase, glucocerebrosidase, lysosomal acid lipase,lysosomal enzyme acid sphingomyelinase, formylglycine-generating enzyme,iduronidase (e.g., alpha-L), acetyl-CoA:alpha-glucosaminideN-acetyltransferase, glycosaminoglycan alpha-L-iduronohydrolase, heparanN-sulfatase, N-acetyl-α-D-glucosaminidase (NAGLU),iduronate-2-sulfatase, galactosamine-6-sulfate sulfatase,N-acetylgalactosamine-6-sulfatase, glycosaminoglycanN-acetylgalactosamine 4-sulfatase, β-glucuronidase, hyaluronidase,alpha-N-acetyl neuraminidase (sialidase), ganglioside sialidase,phosphotransferase, alpha-glucosidase, alpha-D-mannosidase,beta-D-mannosidase, aspartylglucosaminidase, alpha-L-fucosidase,battenin, palmitoyl protein thioesterases, and other Batten-relatedproteins (e.g., ceroid-lipofuscinosis neuronal protein 6), or anenzymatically active fragment thereof. In some embodiments, thetherapeutic protein comprises alpha-glucosidase, or an enzymaticallyactive fragment thereof. In some embodiments, the therapeutic protein isN-acetyl-α-D-glucosaminidase (NAGLU). In some embodiments, the fusionprotein is encoded by a nucleic acid comprising a Kozak sequence.

In additional aspects, there are provided fusion proteins comprising asignal peptide and a therapeutic protein, wherein the signal peptide isremoved after translation upon secretion from the cell. In someembodiments, the signal peptide comprises a binding immunoglobulinprotein (BiP) signal peptide. In some embodiments, the fusion proteinfurther comprises a linker. In some embodiments, the linker consists of5-20 amino acids, 5-15 amino acids, 5-10 amino acids, 8-12 amino acids,or about 7, 8, 9, 10, 11, 12 or 13 amino acids. In some embodiments, thelinker comprises an amino acid sequence of GGGGSGGGG (SEQ ID NO: 18),GGGGS (SEQ ID NO: 19), GGGSGGGGS (SEQ ID NO: 20), GGGGSGGGS (SEQ ID NO:21), GGGGSGGGGS (SEQ ID NO: 37), or GGSGSGSTS (SEQ ID NO: 33). In someembodiments, the fusion protein further comprises a variant IGF2peptide. In some embodiments, the fusion protein is encoded by a nucleicacid comprising a cricket paralysis virus internal ribosome entrysequence (CrPV IRES). In some embodiments, the therapeutic proteincomprises at least one enzyme of the group consisting ofalpha-galactosidase (A or B), β-galactosidase, β-hexosaminidase (A orB), galactosylceramidase, arylsulfatase (A or B), β-glucocerebrosidase,glucocerebrosidase, lysosomal acid lipase, lysosomal enzyme acidsphingomyelinase, formylglycine-generating enzyme, iduronidase (e.g.,alpha-L), acetyl-CoA:alpha-glucosaminide N-acetyltransferase,glycosaminoglycan alpha-L-iduronohydrolase, heparan N-sulfatase,N-acetyl-α-D-glucosaminidase (NAGLU), iduronate-2-sulfatase,galactosamine-6-sulfate sulfatase, N-acetylgalactosamine-6-sulfatase,glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β-glucuronidase,hyaluronidase, alpha-N-acetyl neuraminidase (sialidase), gangliosidesialidase, phosphotransferase, alpha-glucosidase, alpha-D-mannosidase,beta-D-mannosidase, aspartylglucosaminidase, alpha-L-fucosidase,battenin, palmitoyl protein thioesterases, and other Batten-relatedproteins (e.g., ceroid-lipofuscinosis neuronal protein 6), or anenzymatically active fragment thereof. In some embodiments, thetherapeutic protein comprises alpha-glucosidase, or an enzymaticallyactive fragment thereof. In some embodiments, the therapeutic protein isN-acetyl-α-D-glucosaminidase (NAGLU). In some embodiments, the fusionprotein is encoded by a nucleic acid comprising a Kozak sequence.

In further aspects, there are provided nucleic acid sequences encodingfusion proteins comprising a therapeutic protein, wherein the fusionprotein is encoded by a nucleic acid comprising a cricket paralysisvirus internal ribosome entry sequence (CrPV IRES). In some embodiments,the fusion protein further comprises a signal peptide. In someembodiments, the signal peptide comprises a binding immunoglobulinprotein (BiP) signal peptide. In some embodiments, the fusion proteinfurther comprises a variant IGF2 peptide. In some embodiments, thefusion protein further comprises a linker. In some embodiments, thelinker consists of 5-20 amino acids, 5-15 amino acids, 5-10 amino acids,8-12 amino acids, or about 7, 8, 9, 10, 11, 12 or 13 amino acids. Insome embodiments, the linker comprises an amino acid sequence ofGGGGSGGGG (SEQ ID NO: 18), GGGGS (SEQ ID NO: 19), GGGSGGGGS (SEQ ID NO:20), GGGGSGGGS (SEQ ID NO: 21), GGGGSGGGGS (SEQ ID NO: 37), or GGSGSGSTS(SEQ ID NO: 33) In some embodiments, the therapeutic protein comprisesat least one enzyme of the group consisting of alpha-galactosidase (A orB), β-galactosidase, β-hexosaminidase (A or B), galactosylceramidase,arylsulfatase (A or B), β-glucocerebrosidase, glucocerebrosidase,lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase,formylglycine-generating enzyme, iduronidase (e.g., alpha-L),acetyl-CoA:alpha-glucosaminide N-acetyltransferase, glycosaminoglycanalpha-L-iduronohydrolase, heparan N-sulfatase,N-acetyl-α-D-glucosaminidase (NAGLU), iduronate-2-sulfatase,galactosamine-6-sulfate sulfatase, N-acetylgalactosamine-6-sulfatase,glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β-glucuronidase,hyaluronidase, alpha-N-acetyl neuraminidase (sialidase), gangliosidesialidase, phosphotransferase, alpha-glucosidase, alpha-D-mannosidase,beta-D-mannosidase, aspartylglucosaminidase, alpha-L-fucosidase,battenin, palmitoyl protein thioesterases, and other Batten-relatedproteins (e.g., ceroid-lipofuscinosis neuronal protein 6), or anenzymatically active fragment thereof. In some embodiments, thetherapeutic protein comprises alpha-glucosidase, or an enzymaticallyactive fragment thereof. In some embodiments, the therapeutic protein isN-acetyl-α-D-glucosaminidase (NAGLU). In some embodiments, the fusionprotein is encoded by a nucleic acid comprising a Kozak sequence.

In additional aspects, there are provided fusion proteins comprising atherapeutic protein, wherein the fusion protein is encoded by a nucleicacid comprising a Kozak sequence. In some embodiments, the fusionprotein further comprises a linker. In some embodiments, the linkerconsists of 5-20 amino acids, 5-15 amino acids, 5-10 amino acids, 8-12amino acids, or about 7, 8, 9, 10, 11, 12 or 13 amino acids. In someembodiments, the linker comprises an amino acid sequence of GGGGSGGGG(SEQ ID NO: 18), GGGGS (SEQ ID NO: 19), GGGSGGGGS (SEQ ID NO: 20),GGGGSGGGS (SEQ ID NO: 21), or GGSGSGSTS (SEQ ID NO: 33). In someembodiments, the fusion protein further comprises a signal peptide. Insome embodiments, the signal peptide comprises a binding immunoglobulinprotein (BiP) signal peptide. In some embodiments, the fusion proteinfurther comprises a variant IGF2 peptide. In some embodiments, thetherapeutic protein comprises at least one enzyme of the groupconsisting of alpha-galactosidase (A or B), β-galactosidase,β-hexosaminidase (A or B), galactosylceramidase, arylsulfatase (A or B),β-glucocerebrosidase, glucocerebrosidase, lysosomal acid lipase,lysosomal enzyme acid sphingomyelinase, formylglycine-generating enzyme,iduronidase (e.g., alpha-L), acetyl-CoA:alpha-glucosaminideN-acetyltransferase, glycosaminoglycan alpha-L-iduronohydrolase, heparanN-sulfatase, N-acetyl-α-D-glucosaminidase (NAGLU),iduronate-2-sulfatase, galactosamine-6-sulfate sulfatase,N-acetylgalactosamine-6-sulfatase, glycosaminoglycanN-acetylgalactosamine 4-sulfatase, glucuronidase, hyaluronidase,alpha-N-acetyl neuraminidase (sialidase), ganglioside sialidase,phosphotransferase, alpha-glucosidase, alpha-D-mannosidase,beta-D-mannosidase, aspartylglucosaminidase, alpha-L-fucosidase,battenin, palmitoyl protein thioesterases, and other Batten-relatedproteins (e.g., ceroid-lipofuscinosis neuronal protein 6), or anenzymatically active fragment thereof. In some embodiments, thetherapeutic protein comprises alpha-glucosidase, or an enzymaticallyactive fragment thereof. In some embodiments, the therapeutic protein isN-acetyl-α-D-glucosaminidase (NAGLU). In some embodiments, the fusionprotein is encoded by a nucleic acid comprising a cricket paralysisvirus internal ribosome entry sequence (CrPV IBES).

In additional aspects, there are provided nucleic acids encoding afusion protein, such as nucleic acids encoding a fusion proteincomprising a variant IGF2 peptide and a therapeutic protein. In someembodiments, the fusion protein further comprises a linker. In someembodiments, the linker consists of 5-20 amino acids, 5-15 amino acids,5-10 amino acids, 8-12 amino acids, or about 7, 8, 9, 10, 11, 12 or 13amino acids. In some embodiments, the linker comprises an amino acidsequence of GGGGSGGGG (SEQ ID NO: 18), GGGGS (SEQ ID NO: 19), GGGSGGGGS(SEQ ID NO: 20), GGGGSGGGS (SEQ ID NO: 21), GGGGSGGGGS (SEQ ID NO: 37),or GGSGSGSTS (SEQ ID NO: 33). In some embodiments, the fusion proteinfurther comprises a signal peptide. In some embodiments, the signalpeptide comprises a binding immunoglobulin protein (BiP) signal peptide.In some embodiments, the nucleic acid further comprises a cricketparalysis virus internal ribosome entry sequence (CrPV IBES). In someembodiments, the nucleic acid further comprises a Kozak sequence. Insome embodiments, the therapeutic protein comprises at least one enzymeof the group consisting of alpha-galactosidase (A or B),β-galactosidase, β-hexosaminidase (A or B), galactosylceramidase,arylsulfatase (A or B), β-glucocerebrosidase, glucocerebrosidase,lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase,formylglycine-generating enzyme, iduronidase (e.g., alpha-L),acetyl-CoA:alpha-glucosaminide N-acetyltransferase, glycosaminoglycanalpha-L-iduronohydrolase, heparan N-sulfatase,N-acetyl-α-D-glucosaminidase (NAGLU), iduronate-2-sulfatase,galactosamine-6-sulfate sulfatase, N-acetylgalactosamine-6-sulfatase,glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β-glucuronidase,hyaluronidase, alpha-N-acetyl neuraminidase (sialidase), gangliosidesialidase, phosphotransferase, alpha-glucosidase, alpha-D-mannosidase,beta-D-mannosidase, aspartylglucosaminidase, alpha-L-fucosidase,battenin, palmitoyl protein thioesterases, and other Batten-relatedproteins (e.g., ceroid-lipofuscinosis neuronal protein 6), or anenzymatically active fragment thereof. In some embodiments, thetherapeutic protein comprises alpha-glucosidase, or an enzymaticallyactive fragment thereof. In some embodiments, the therapeutic protein isN-acetyl-α-D-glucosaminidase (NAGLU). In some embodiments, the nucleicacid further comprises a promoter. In some embodiments, the nucleic acidis comprised within a viral vector. In some embodiments, the viralvector comprises a retrovirus, an adenovirus, an adeno associated virus,a lentivirus, or a herpes virus.

In further aspects, there are provided nucleic acids encoding a fusionprotein comprising a signal peptide and a therapeutic protein. In someembodiments, the signal peptide comprises a binding immunoglobulinprotein (BiP) signal peptide. In some embodiments, the fusion proteinfurther comprises a linker. In some embodiments, the linker consists of5-20 amino acids, 5-15 amino acids, 5-10 amino acids, 8-12 amino acids,or about 7, 8, 9, 10, 11, 12 or 13 amino acids. In some embodiments, thelinker comprises an amino acid sequence of GGGGSGGGG (SEQ ID NO: 18),GGGGS (SEQ ID NO: 19), GGGSGGGGS (SEQ ID NO: 20), GGGGSGGGS (SEQ ID NO:21), GGGGSGGGGS (SEQ ID NO: 37), or GGSGSGSTS (SEQ ID NO: 33). In someembodiments, the fusion protein further comprises a variant IGF2peptide. In some embodiments, the nucleic acid further comprises acricket paralysis virus internal ribosome entry sequence (CrPV IRES). Insome embodiments, the nucleic acid further comprises a Kozak sequence.In some embodiments, the therapeutic protein comprises at least oneenzyme of the group consisting of alpha-galactosidase (A or B),β-galactosidase, β-hexosaminidase (A or B), galactosylceramidase,arylsulfatase (A or B), β-glucocerebrosidase, glucocerebrosidase,lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase,formylglycine-generating enzyme, iduronidase (e.g., alpha-L),acetyl-CoA:alpha-glucosaminide N-acetyltransferase, glycosaminoglycanalpha-L-iduronohydrolase, heparan N-sulfatase,N-acetyl-α-D-glucosaminidase (NAGLU), iduronate-2-sulfatase,galactosamine-6-sulfate sulfatase, N-acetylgalactosamine-6-sulfatase,glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β-glucuronidase,hyaluronidase, alpha-N-acetyl neuraminidase (sialidase), gangliosidesialidase, phosphotransferase, alpha-glucosidase, alpha-D-mannosidase,beta-D-mannosidase, aspartylglucosaminidase, alpha-L-fucosidase,battenin, palmitoyl protein thioesterases, and other Batten-relatedproteins (e.g., ceroid-lipofuscinosis neuronal protein 6), or anenzymatically active fragment thereof. In some embodiments, thetherapeutic protein comprises alpha-glucosidase, or an enzymaticallyactive fragment thereof. In some embodiments, the therapeutic protein isN-acetyl-α-D-glucosaminidase (NAGLU). In some embodiments, the nucleicacid further comprises a promoter. In some embodiments, the nucleic acidis comprised within a viral vector. In some embodiments, the viralvector comprises a retrovirus, an adenovirus, an adeno associated virus,a lentivirus, or a herpes virus.

In additional aspects, there are provided nucleic acids encoding afusion protein comprising a therapeutic protein, wherein the nucleicacid further comprises a cricket paralysis virus internal ribosome entrysequence (CrPV IRES). In some embodiments, the nucleic acid furthercomprises a Kozak sequence. In some embodiments, the fusion proteinfurther comprises a linker. In some embodiments, the linker consists of5-20 amino acids, 5-15 amino acids, 5-10 amino acids, 8-12 amino acids,or about 7, 8, 9, 10, 11, 12 or 13 amino acids. In some embodiments, thelinker comprises an amino acid sequence of GGGGSGGGG (SEQ ID NO: 18),GGGGS (SEQ ID NO: 19), GGGSGGGGS (SEQ ID NO: 20), GGGGSGGGS (SEQ ID NO:21), GGGGSGGGGS (SEQ ID NO: 37), or GGSGSGSTS (SEQ ID NO: 33). In someembodiments, the fusion protein further comprises a signal peptide. Insome embodiments, the signal peptide comprises a binding immunoglobulinprotein (BiP) signal peptide. In some embodiments, the fusion proteinfurther comprises a variant IGF2 peptide. In some embodiments, thetherapeutic protein comprises at least one enzyme of the groupconsisting of alpha-galactosidase (A or B), β-galactosidase,β-hexosaminidase (A or B), galactosylceramidase, arylsulfatase (A or B),β-glucocerebrosidase, glucocerebrosidase, lysosomal acid lipase,lysosomal enzyme acid sphingomyelinase, formylglycine-generating enzyme,iduronidase (e.g., alpha-L), acetyl-CoA:alpha-glucosaminideN-acetyltransferase, glycosaminoglycan alpha-L-iduronohydrolase, heparanN-sulfatase, N-acetyl-α-D-glucosaminidase (NAGLU),iduronate-2-sulfatase, galactosamine-6-sulfate sulfatase,N-acetylgalactosamine-6-sulfatase, glycosaminoglycanN-acetylgalactosamine 4-sulfatase, β-glucuronidase, hyaluronidase,alpha-N-acetyl neuraminidase (sialidase), ganglioside sialidase,phosphotransferase, alpha-glucosidase, alpha-D-mannosidase,beta-D-mannosidase, aspartylglucosaminidase, alpha-L-fucosidase,battenin, palmitoyl protein thioesterases, and other Batten-relatedproteins (e.g., ceroid-lipofuscinosis neuronal protein 6), or anenzymatically active fragment thereof. In some embodiments, thetherapeutic protein comprises alpha-glucosidase, or an enzymaticallyactive fragment thereof. In some embodiments, the therapeutic protein isN-acetyl-α-D-glucosaminidase (NAGLU). In some embodiments, the nucleicacid further comprises a promoter. In some embodiments, the nucleic acidis comprised within a viral vector. In some embodiments, the viralvector comprises a retrovirus, an adenovirus, an adeno associated virus,a lentivirus, or a herpes virus.

In further aspects, there are provided nucleic acids encoding a fusionprotein comprising a therapeutic protein, wherein the nucleic acidfurther comprises a Kozak sequence. In some embodiments, the nucleicacid further comprises a Kozak sequence. In some embodiments, the fusionprotein further comprises a linker. In some embodiments, the linkerconsists of 5-20 amino acids, 5-15 amino acids, 5-10 amino acids, 8-12amino acids, or about 7, 8, 9, 10, 11, 12 or 13 amino acids. In someembodiments, the linker comprises an amino acid sequence of GGGGSGGGG(SEQ ID NO: 18), GGGGS (SEQ ID NO: 19), GGGSGGGGS (SEQ ID NO: 20),GGGGSGGGS (SEQ ID NO: 21), GGGGSGGGGS (SEQ ID NO: 37), or GGSGSGSTS (SEQID NO: 33). In some embodiments, the fusion protein further comprises asignal peptide. In some embodiments, the signal peptide comprises abinding immunoglobulin protein (BiP) signal peptide. In someembodiments, the fusion protein further comprises a variant IGF2peptide. In some embodiments, the therapeutic protein comprises at leastone enzyme of the group consisting of alpha-galactosidase (A or B),β-galactosidase, β-hexosaminidase (A or B), galactosylceramidase,arylsulfatase (A or B), β-glucocerebrosidase, glucocerebrosidase,lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase,formylglycine-generating enzyme, iduronidase (e.g., alpha-L),acetyl-CoA:alpha-glucosaminide N-acetyltransferase, glycosaminoglycanalpha-L-iduronohydrolase, heparan N-sulfatase,N-acetyl-α-D-glucosaminidase (NAGLU), iduronate-2-sulfatase,galactosamine-6-sulfate sulfatase, N-acetylgalactosamine-6-sulfatase,glycosaminoglycan N-acetylgalactosamine 4-sulfatase, glucuronidase,hyaluronidase, alpha-N-acetyl neuraminidase (sialidase), gangliosidesialidase, phosphotransferase, alpha-glucosidase, alpha-D-mannosidase,beta-D-mannosidase, aspartylglucosaminidase, alpha-L-fucosidase,battenin, palmitoyl protein thioesterases, and other Batten-relatedproteins (e.g., ceroid-lipofuscinosis neuronal protein 6), or anenzymatically active fragment thereof. In some embodiments, thetherapeutic protein comprises alpha-glucosidase, or an enzymaticallyactive fragment thereof. In some embodiments, the therapeutic protein isN-acetyl-α-D-glucosaminidase (NAGLU). In some embodiments, the nucleicacid further comprises a promoter. In some embodiments, the nucleic acidis comprised within a viral vector. In some embodiments, the viralvector comprises a retrovirus, an adenovirus, an adeno associated virus,a lentivirus, or a herpes virus.

In additional aspects, there are provided compositions comprising (a) anucleic acid encoding a fusion protein comprising a variant IGF2 peptideand a therapeutic protein; and (b) a buffer or excipient suitable forgene therapy. In some embodiments, the fusion protein further comprisesa linker. In some embodiments, the linker consists of 5-20 amino acids,5-15 amino acids, 5-10 amino acids, 8-12 amino acids, or about 7, 8, 9,10, 11, 12 or 13 amino acids. In some embodiments, the linker comprisesan amino acid sequence of GGGGSGGGG (SEQ ID NO: 18), GGGGS (SEQ ID NO:19), GGGSGGGGS (SEQ ID NO: 20), GGGGSGGGS (SEQ ID NO: 21), GGGGSGGGGS(SEQ ID NO: 37), or GGSGSGSTS (SEQ ID NO: 33). In some embodiments, thefusion protein further comprises a signal peptide. In some embodiments,the signal peptide comprises a binding immunoglobulin protein (BiP)signal peptide. In some embodiments, the nucleic acid further comprisesa cricket paralysis virus internal ribosome entry sequence (CrPV IBES).In some embodiments, the nucleic acid further comprises a Kozaksequence. In some embodiments, the therapeutic protein comprises atleast one enzyme of the group consisting of alpha-galactosidase (A orB), β-galactosidase, β-hexosaminidase (A or B), galactosylceramidase,arylsulfatase (A or B), β-glucocerebrosidase, glucocerebrosidase,lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase,formylglycine-generating enzyme, iduronidase (e.g., alpha-L),acetyl-CoA:alpha-glucosaminide N-acetyltransferase, glycosaminoglycanalpha-L-iduronohydrolase, heparan N-sulfatase,N-acetyl-α-D-glucosaminidase (NAGLU), iduronate-2-sulfatase,galactosamine-6-sulfate sulfatase, N-acetylgalactosamine-6-sulfatase,glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β-glucuronidase,hyaluronidase, alpha-N-acetyl neuraminidase (sialidase), gangliosidesialidase, phosphotransferase, alpha-glucosidase, alpha-D-mannosidase,beta-D-mannosidase, aspartylglucosaminidase, alpha-L-fucosidase,battenin, palmitoyl protein thioesterases, and other Batten-relatedproteins (e.g., ceroid-lipofuscinosis neuronal protein 6), or anenzymatically active fragment thereof. In some embodiments, thetherapeutic protein comprises alpha-glucosidase, or an enzymaticallyactive fragment thereof. In some embodiments, the therapeutic protein isN-acetyl-α-D-glucosaminidase (NAGLU). In some embodiments, the nucleicacid further comprises a promoter. In some embodiments, the nucleic acidis comprised within a viral vector. In some embodiments, the viralvector comprises a retrovirus, an adenovirus, an adeno associated virus,a lentivirus, or a herpes virus. In some embodiments, the buffer orexcipient suitable for gene therapy comprises a liposome, ananoparticle, or a cell-penetrating peptide. In some embodiments, thebuffer or excipient suitable for gene therapy comprises a viral coatprotein. In some embodiments, the viral coat protein is selected fromthe group consisting of a vesicular stomatitis virus coat protein, anadenovirus coat protein, an adeno-associated virus coat protein, amurine leukemia virus coat protein, an HIV coat protein, and aninfluenza virus coat protein.

In additional aspects, there are provided compositions comprising (a) anucleic acid encoding a fusion protein comprising a signal peptide and atherapeutic protein; and (b) a buffer or excipient suitable for genetherapy. In some embodiments, the signal peptide comprises a bindingimmunoglobulin protein (BiP) signal peptide. In some embodiments, thefusion protein further comprises a variant IGF2 peptide. In someembodiments, the fusion protein further comprises a linker. In someembodiments, the linker consists of 5-20 amino acids, 5-15 amino acids,5-10 amino acids, 8-12 amino acids, or about 7, 8, 9, 10, 11, 12 or 13amino acids. In some embodiments, the linker comprises an amino acidsequence of GGGGSGGGG (SEQ ID NO: 18), GGGGS (SEQ ID NO: 19), GGGSGGGGS(SEQ ID NO: 20), GGGGSGGGS (SEQ ID NO: 21), GGGGSGGGGS (SEQ ID NO: 37),or GGSGSGSTS (SEQ ID NO: 33). In some embodiments, the nucleic acidfurther comprises a cricket paralysis virus internal ribosome entrysequence (CrPV IBES). In some embodiments, the nucleic acid furthercomprises a Kozak sequence. In some embodiments, the therapeutic proteincomprises at least one enzyme of the group consisting ofalpha-galactosidase (A or B), β-galactosidase, β-hexosaminidase (A orB), galactosylceramidase, arylsulfatase (A or B), β-glucocerebrosidase,glucocerebrosidase, lysosomal acid lipase, lysosomal enzyme acidsphingomyelinase, formylglycine-generating enzyme, iduronidase (e.g.,alpha-L), acetyl-CoA:alpha-glucosaminide N-acetyltransferase,glycosaminoglycan alpha-L-iduronohydrolase, heparan N-sulfatase,N-acetyl-α-D-glucosaminidase (NAGLU), iduronate-2-sulfatase,galactosamine-6-sulfate sulfatase, N-acetylgalactosamine-6-sulfatase,glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β-glucuronidase,hyaluronidase, alpha-N-acetyl neuraminidase (sialidase), gangliosidesialidase, phosphotransferase, alpha-glucosidase, alpha-D-mannosidase,beta-D-mannosidase, aspartylglucosaminidase, alpha-L-fucosidase,battenin, palmitoyl protein thioesterases, and other Batten-relatedproteins (e.g., ceroid-lipofuscinosis neuronal protein 6), or anenzymatically active fragment thereof. In some embodiments, thetherapeutic protein comprises alpha-glucosidase, or an enzymaticallyactive fragment thereof. In some embodiments, the therapeutic protein isN-acetyl-α-D-glucosaminidase (NAGLU). In some embodiments, the nucleicacid further comprises a promoter. In some embodiments, the nucleic acidis comprised within a viral vector. In some embodiments, the viralvector comprises a retrovirus, an adenovirus, an adeno associated virus,a lentivirus, or a herpes virus. In some embodiments, the buffer orexcipient suitable for gene therapy comprises a liposome, ananoparticle, or a cell-penetrating peptide. In some embodiments, thebuffer or excipient suitable for gene therapy comprises a viral coatprotein. In some embodiments, the viral coat protein is selected fromthe group consisting of a vesicular stomatitis virus coat protein, anadenovirus coat protein, an adeno-associated virus coat protein, amurine leukemia virus coat protein, an HIV coat protein, and aninfluenza virus coat protein.

In further aspects, there are provided compositions comprising (a) anucleic acid encoding a fusion protein comprising a therapeutic protein;and (b) a buffer or excipient suitable for gene therapy, wherein thenucleic acid further comprises a cricket paralysis virus internalribosome entry sequence (CrPV IBES). In some embodiments, the nucleicacid further comprises a Kozak sequence. In some embodiments, the fusionprotein further comprises a linker. In some embodiments, the linkerconsists of 5-20 amino acids, 5-15 amino acids, 5-10 amino acids, 8-12amino acids, or about 7, 8, 9, 10, 11, 12 or 13 amino acids. In someembodiments, the linker comprises an amino acid sequence of GGGGSGGGG(SEQ ID NO: 18), GGGGS (SEQ ID NO: 19), GGGSGGGGS (SEQ ID NO: 20),GGGGSGGGS (SEQ ID NO: 21), GGGGSGGGGS (SEQ ID NO: 37), or GGSGSGSTS (SEQID NO: 33). In some embodiments, the fusion protein further comprises asignal peptide. In some embodiments, the signal peptide comprises abinding immunoglobulin protein (BiP) signal peptide. In someembodiments, the fusion protein further comprises a variant IGF2peptide. In some embodiments, the therapeutic protein comprises at leastone enzyme of the group consisting of alpha-galactosidase (A or B),β-galactosidase, β-hexosaminidase (A or B), galactosylceramidase,arylsulfatase (A or B), β-glucocerebrosidase, glucocerebrosidase,lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase,formylglycine-generating enzyme, iduronidase (e.g., alpha-L),acetyl-CoA:alpha-glucosaminide N-acetyltransferase, glycosaminoglycanalpha-L-iduronohydrolase, heparan N-sulfatase,N-acetyl-α-D-glucosaminidase (NAGLU), iduronate-2-sulfatase,galactosamine-6-sulfate sulfatase, N-acetylgalactosamine-6-sulfatase,glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β-glucuronidase,hyaluronidase, alpha-N-acetyl neuraminidase (sialidase), gangliosidesialidase, phosphotransferase, alpha-glucosidase, alpha-D-mannosidase,beta-D-mannosidase, aspartylglucosaminidase, alpha-L-fucosidase,battenin, palmitoyl protein thioesterases, and other Batten-relatedproteins (e.g., ceroid-lipofuscinosis neuronal protein 6), or anenzymatically active fragment thereof. In some embodiments, thetherapeutic protein comprises alpha-glucosidase, or an enzymaticallyactive fragment thereof. In some embodiments, the therapeutic protein isN-acetyl-α-D-glucosaminidase (NAGLU). In some embodiments, the nucleicacid further comprises a promoter. In some embodiments, the nucleic acidis comprised within a viral vector. In some embodiments, the viralvector comprises a retrovirus, an adenovirus, an adeno associated virus,a lentivirus, or a herpes virus. In some embodiments, the buffer orexcipient suitable for gene therapy comprises a liposome, ananoparticle, or a cell-penetrating peptide. In some embodiments, thebuffer or excipient suitable for gene therapy comprises a viral coatprotein. In some embodiments, the viral coat protein is selected fromthe group consisting of a vesicular stomatitis virus coat protein, anadenovirus coat protein, an adeno-associated virus coat protein, amurine leukemia virus coat protein, an HIV coat protein, and aninfluenza virus coat protein.

In further aspects, there are provided compositions comprising (a) anucleic acid encoding a fusion protein comprising a therapeutic protein;and (b) a buffer or excipient suitable for gene therapy, wherein thenucleic acid further comprises a Kozak sequence. In some embodiments,the nucleic acid further comprises a cricket paralysis virus internalribosome entry sequence (CrPV IBES). In some embodiments, the fusionprotein further comprises a linker. In some embodiments, the linkerconsists of 5-20 amino acids, 5-15 amino acids, 5-10 amino acids, 8-12amino acids, or about 7, 8, 9, 10, 11, 12 or 13 amino acids. In someembodiments, the linker comprises an amino acid sequence of GGGGSGGGG(SEQ ID NO: 18), GGGGS (SEQ ID NO: 19), GGGSGGGGS (SEQ ID NO: 20),GGGGSGGGS (SEQ ID NO: 21), GGGGSGGGGS (SEQ ID NO: 37), or GGSGSGSTS (SEQID NO: 33). In some embodiments, the fusion protein further comprises asignal peptide. In some embodiments, the signal peptide comprises abinding immunoglobulin protein (BiP) signal peptide. In someembodiments, the fusion protein further comprises a variant IGF2peptide. In some embodiments, the therapeutic protein comprises at leastone enzyme of the group consisting of alpha-galactosidase (A or B),β-galactosidase, β-hexosaminidase (A or B), galactosylceramidase,arylsulfatase (A or B), β-glucocerebrosidase, glucocerebrosidase,lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase,formylglycine-generating enzyme, iduronidase (e.g., alpha-L),acetyl-CoA:alpha-glucosaminide N-acetyltransferase, glycosaminoglycanalpha-L-iduronohydrolase, heparan N-sulfatase,N-acetyl-α-D-glucosaminidase (NAGLU), iduronate-2-sulfatase,galactosamine-6-sulfate sulfatase, N-acetylgalactosamine-6-sulfatase,glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β-glucuronidase,hyaluronidase, alpha-N-acetyl neuraminidase (sialidase), gangliosidesialidase, phosphotransferase, alpha-glucosidase, alpha-D-mannosidase,beta-D-mannosidase, aspartylglucosaminidase, alpha-L-fucosidase,battenin, palmitoyl protein thioesterases, and other Batten-relatedproteins (e.g., ceroid-lipofuscinosis neuronal protein 6), or anenzymatically active fragment thereof. In some embodiments, thetherapeutic protein comprises alpha-glucosidase, or an enzymaticallyactive fragment thereof. In some embodiments, the therapeutic protein isN-acetyl-α-D-glucosaminidase (NAGLU). In some embodiments, the nucleicacid further comprises a promoter. In some embodiments, the nucleic acidis comprised within a viral vector. In some embodiments, the viralvector comprises a retrovirus, an adenovirus, an adeno associated virus,a lentivirus, or a herpes virus. In some embodiments, the buffer orexcipient suitable for gene therapy comprises a liposome, ananoparticle, or a cell-penetrating peptide. In some embodiments, thebuffer or excipient suitable for gene therapy comprises a viral coatprotein. In some embodiments, the viral coat protein is selected fromthe group consisting of a vesicular stomatitis virus coat protein, anadenovirus coat protein, an adeno-associated virus coat protein, amurine leukemia virus coat protein, an HIV coat protein, and aninfluenza virus coat protein.

In additional aspects, there are provided methods of treating a geneticdisorder in an individual comprising administering a compositioncomprising (a) a nucleic acid encoding a fusion protein comprising avariant IGF2 peptide and a therapeutic protein; and (b) a buffer orexcipient suitable for gene therapy. In some embodiments, the fusionprotein further comprises a linker. In some embodiments, the linkerconsists of 5-20 amino acids, 5-15 amino acids, 5-10 amino acids, 8-12amino acids, or about 7, 8, 9, 10, 11, 12 or 13 amino acids. In someembodiments, the linker comprises an amino acid sequence of GGGGSGGGG(SEQ ID NO: 18), GGGGS (SEQ ID NO: 19), GGGSGGGGS (SEQ ID NO: 20),GGGGSGGGS (SEQ ID NO: 21), GGGGSGGGGS (SEQ ID NO: 37), or GGSGSGSTS (SEQID NO: 33). In some embodiments, the fusion protein further comprises asignal peptide. In some embodiments, the signal peptide comprises abinding immunoglobulin protein (BiP) signal peptide. In someembodiments, the nucleic acid further comprises a cricket paralysisvirus internal ribosome entry sequence (CrPV IBES). In some embodiments,the nucleic acid further comprises a Kozak sequence. In someembodiments, the therapeutic protein comprises at least one enzyme ofthe group consisting of alpha-galactosidase (A or B), β-galactosidase,β-hexosaminidase (A or B), galactosylceramidase, arylsulfatase (A or B),β-glucocerebrosidase, glucocerebrosidase, lysosomal acid lipase,lysosomal enzyme acid sphingomyelinase, formylglycine-generating enzyme,iduronidase (e.g., alpha-L), acetyl-CoA:alpha-glucosaminideN-acetyltransferase, glycosaminoglycan alpha-L-iduronohydrolase, heparanN-sulfatase, N-acetyl-α-D-glucosaminidase (NAGLU),iduronate-2-sulfatase, galactosamine-6-sulfate sulfatase,N-acetylgalactosamine-6-sulfatase, glycosaminoglycanN-acetylgalactosamine 4-sulfatase, β-glucuronidase, hyaluronidase,alpha-N-acetyl neuraminidase (sialidase), ganglioside sialidase,phosphotransferase, alpha-glucosidase, alpha-D-mannosidase,beta-D-mannosidase, aspartylglucosaminidase, alpha-L-fucosidase,battenin, palmitoyl protein thioesterases, and other Batten-relatedproteins (e.g., ceroid-lipofuscinosis neuronal protein 6), or anenzymatically active fragment thereof. In some embodiments, thetherapeutic protein comprises alpha-glucosidase, or an enzymaticallyactive fragment thereof. In some embodiments, the therapeutic protein isN-acetyl-α-D-glucosaminidase (NAGLU). In some embodiments, the nucleicacid further comprises a promoter. In some embodiments, the nucleic acidis comprised within a viral vector. In some embodiments, the viralvector comprises a retrovirus, an adenovirus, an adeno associated virus,a lentivirus, or a herpes virus. In some embodiments, the buffer orexcipient suitable for gene therapy comprises a liposome, ananoparticle, or a cell-penetrating peptide. In some embodiments, thebuffer or excipient suitable for gene therapy comprises a viral coatprotein. In some embodiments, the viral coat protein is selected fromthe group consisting of a vesicular stomatitis virus coat protein, anadenovirus coat protein, an adeno-associated virus coat protein, amurine leukemia virus coat protein, an HIV coat protein, and aninfluenza virus coat protein. In some embodiments, the genetic disorderis a lysosomal storage disorder. In some embodiments, the geneticdisorder is selected from the group consisting ofaspartylglucosaminuria, Batten disease, cystinosis, Fabry disease,Gaucher disease type I, Gaucher disease type II, Gaucher disease typeIII, Pompe disease, Tay Sachs disease, Sandhoff disease, metachomaticleukodystrophy, mucolipidosis type I, mucolipidosis type II,mucolipidosis type III, mucolipidosis type IV, Hurler disease, Hunterdisease, Sanfilippo disease type A, Sanfilippo disease type B,Sanfilippo disease type C, Sanfilippo disease type D, Morquio diseasetype A, Morquio disease type B, Maroteau-Lamy disease, Sly disease,Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pickdisease type C1, Niemann-Pick disease type C2, Schindler disease type I,Schindler disease type II, adenosine deaminase severe combinedimmunodeficiency (ADA-SCID), and chronic granulomatous disease (CGD). Insome embodiments, cells from the individual are treated ex vivo andadministered to the individual after ex vivo treatment.

In additional aspects, there are provided methods of treating a geneticdisorder in an individual comprising administering a compositioncomprising (a) a nucleic acid encoding a fusion protein comprising asignal peptide and a therapeutic protein; and (b) a buffer or excipientsuitable for gene therapy. In some embodiments, the signal peptidecomprises a binding immunoglobulin protein (BiP) signal peptide. In someembodiments, the fusion protein further comprises a linker. In someembodiments, the linker consists of 5-20 amino acids, 5-15 amino acids,5-10 amino acids, 8-12 amino acids, or about 7, 8, 9, 10, 11, 12 or 13amino acids. In some embodiments, the linker comprises an amino acidsequence of GGGGSGGGG (SEQ ID NO: 18), GGGGS (SEQ ID NO: 19), GGGSGGGGS(SEQ ID NO: 20), GGGGSGGGS (SEQ ID NO: 21), GGGGSGGGGS (SEQ ID NO: 37),or GGSGSGSTS (SEQ ID NO: 33). In some embodiments, the fusion proteinfurther comprises a variant IGF2 peptide. In some embodiments, thenucleic acid further comprises a cricket paralysis virus internalribosome entry sequence (CrPV IBES). In some embodiments, the nucleicacid further comprises a Kozak sequence. In some embodiments, thetherapeutic protein comprises at least one enzyme of the groupconsisting of alpha-galactosidase (A or B), β-galactosidase,β-hexosaminidase (A or B), galactosylceramidase, arylsulfatase (A or B),β-glucocerebrosidase, glucocerebrosidase, lysosomal acid lipase,lysosomal enzyme acid sphingomyelinase, formylglycine-generating enzyme,iduronidase (e.g., alpha-L), acetyl-CoA:alpha-glucosaminideN-acetyltransferase, glycosaminoglycan alpha-L-iduronohydrolase, heparanN-sulfatase, N-acetyl-α-D-glucosaminidase (NAGLU),iduronate-2-sulfatase, galactosamine-6-sulfate sulfatase,N-acetylgalactosamine-6-sulfatase, glycosaminoglycanN-acetylgalactosamine 4-sulfatase, β-glucuronidase, hyaluronidase,alpha-N-acetyl neuraminidase (sialidase), ganglioside sialidase,phosphotransferase, alpha-glucosidase, alpha-D-mannosidase,beta-D-mannosidase, aspartylglucosaminidase, alpha-L-fucosidase,battenin, palmitoyl protein thioesterases, and other Batten-relatedproteins (e.g., ceroid-lipofuscinosis neuronal protein 6), or anenzymatically active fragment thereof. In some embodiments, thetherapeutic protein comprises alpha-glucosidase, or an enzymaticallyactive fragment thereof. In some embodiments, the therapeutic protein isN-acetyl-α-D-glucosaminidase (NAGLU). In some embodiments, the nucleicacid further comprises a promoter. In some embodiments, the nucleic acidis comprised within a viral vector. In some embodiments, the viralvector comprises a retrovirus, an adenovirus, an adeno associated virus,a lentivirus, or a herpes virus. In some embodiments, the buffer orexcipient suitable for gene therapy comprises a liposome, ananoparticle, or a cell-penetrating peptide. In some embodiments, thebuffer or excipient suitable for gene therapy comprises a viral coatprotein. In some embodiments, the viral coat protein is selected fromthe group consisting of a vesicular stomatitis virus coat protein, anadenovirus coat protein, an adeno-associated virus coat protein, amurine leukemia virus coat protein, an HIV coat protein, and aninfluenza virus coat protein. In some embodiments, the genetic disorderis a lysosomal storage disorder. In some embodiments, the geneticdisorder is selected from the group consisting ofaspartylglucosaminuria, Batten disease, cystinosis, Fabry disease,Gaucher disease type I, Gaucher disease type II, Gaucher disease typeIII, Pompe disease, Tay Sachs disease, Sandhoff disease, metachomaticleukodystrophy, mucolipidosis type I, mucolipidosis type II,mucolipidosis type III, mucolipidosis type IV, Hurler disease, Hunterdisease, Sanfilippo disease type A, Sanfilippo disease type B,Sanfilippo disease type C, Sanfilippo disease type D, Morquio diseasetype A, Morquio disease type B, Maroteau-Lamy disease, Sly disease,Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pickdisease type C1, Niemann-Pick disease type C2, Schindler disease type I,Schindler disease type II, adenosine deaminase severe combinedimmunodeficiency (ADA-SCID), and chronic granulomatous disease (CGD). Insome embodiments, cells from the individual are treated ex vivo andadministered to the individual after ex vivo treatment.

In further aspects, there are provided methods of treating a geneticdisorder in an individual comprising administering a compositioncomprising (a) a nucleic acid encoding a fusion protein comprising atherapeutic protein and a targeting peptide; and (b) a buffer orexcipient suitable for gene therapy, wherein the nucleic acid furthercomprises a cricket paralysis virus internal ribosome entry sequence(CrPV IBES). In some embodiments, the nucleic acid further comprises aKozak sequence. In some embodiments, the fusion protein furthercomprises a linker. In some embodiments, the linker consists of 5-20amino acids, 5-15 amino acids, 5-10 amino acids, 8-12 amino acids, orabout 7, 8, 9, 10, 11, 12 or 13 amino acids. In some embodiments, thelinker comprises an amino acid sequence of GGGGSGGGG (SEQ ID NO: 18),GGGGS (SEQ ID NO: 19), GGGSGGGGS (SEQ ID NO: 20), GGGGSGGGS (SEQ ID NO:21), GGGGSGGGGS (SEQ ID NO: 37), or GGSGSGSTS (SEQ ID NO: 33). In someembodiments, the fusion protein further comprises a signal peptide. Insome embodiments, the signal peptide comprises a binding immunoglobulinprotein (BiP) signal peptide. In some embodiments, the fusion proteinfurther comprises a variant IGF2 peptide. In some embodiments, thetherapeutic protein comprises at least one enzyme of the groupconsisting of alpha-galactosidase (A or B), β-galactosidase,β-hexosaminidase (A or B), galactosylceramidase, arylsulfatase (A or B),β-glucocerebrosidase, glucocerebrosidase, lysosomal acid lipase,lysosomal enzyme acid sphingomyelinase, formylglycine-generating enzyme,iduronidase (e.g., alpha-L), acetyl-CoA:alpha-glucosaminideN-acetyltransferase, glycosaminoglycan alpha-L-iduronohydrolase, heparanN-sulfatase, N-acetyl-α-D-glucosaminidase (NAGLU),iduronate-2-sulfatase, galactosamine-6-sulfate sulfatase,N-acetylgalactosamine-6-sulfatase, glycosaminoglycanN-acetylgalactosamine 4-sulfatase, β-glucuronidase, hyaluronidase,alpha-N-acetyl neuraminidase (sialidase), ganglioside sialidase,phosphotransferase, alpha-glucosidase, alpha-D-mannosidase,beta-D-mannosidase, aspartylglucosaminidase, alpha-L-fucosidase,battenin, palmitoyl protein thioesterases, and other Batten-relatedproteins (e.g., ceroid-lipofuscinosis neuronal protein 6), or anenzymatically active fragment thereof. In some embodiments, thetherapeutic protein comprises alpha-glucosidase, or an enzymaticallyactive fragment thereof. In some embodiments, the therapeutic protein isN-acetyl-α-D-glucosaminidase (NAGLU). In some embodiments, the nucleicacid further comprises a promoter. In some embodiments, the nucleic acidis comprised within a viral vector. In some embodiments, the viralvector comprises a retrovirus, an adenovirus, an adeno associated virus,a lentivirus, or a herpes virus. In some embodiments, the buffer orexcipient suitable for gene therapy comprises a liposome, ananoparticle, or a cell-penetrating peptide. In some embodiments, thebuffer or excipient suitable for gene therapy comprises a viral coatprotein. In some embodiments, the viral coat protein is selected fromthe group consisting of a vesicular stomatitis virus coat protein, anadenovirus coat protein, an adeno-associated virus coat protein, amurine leukemia virus coat protein, an HIV coat protein, and aninfluenza virus coat protein. In some embodiments, the genetic disorderis a lysosomal storage disorder. In some embodiments, the geneticdisorder is selected from the group consisting ofaspartylglucosaminuria, Batten disease, cystinosis, Fabry disease,Gaucher disease type I, Gaucher disease type II, Gaucher disease typeIII, Pompe disease, Tay Sachs disease, Sandhoff disease, metachomaticleukodystrophy, mucolipidosis type I, mucolipidosis type II,mucolipidosis type III, mucolipidosis type IV, Hurler disease, Hunterdisease, Sanfilippo disease type A, Sanfilippo disease type B,Sanfilippo disease type C, Sanfilippo disease type D, Morquio diseasetype A, Morquio disease type B, Maroteau-Lamy disease, Sly disease,Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pickdisease type C1, Niemann-Pick disease type C2, Schindler disease type I,Schindler disease type II, adenosine deaminase severe combinedimmunodeficiency (ADA-SCID), and chronic granulomatous disease (CGD). Insome embodiments, cells from the individual are treated ex vivo andadministered to the individual after ex vivo treatment.

In additional aspects, there are provided methods of treating a geneticdisorder in an individual comprising administering a compositioncomprising (a) a nucleic acid encoding a fusion protein comprising atherapeutic protein; and (b) a buffer or excipient suitable for genetherapy, wherein the nucleic acid further comprises a Kozak sequence. Insome embodiments, the nucleic acid further comprises a cricket paralysisvirus internal ribosome entry sequence (CrPV IBES). In some embodiments,the fusion protein further comprises a signal peptide. In someembodiments, the signal peptide comprises a binding immunoglobulinprotein (BiP) signal peptide. In some embodiments, the fusion proteinfurther comprises a variant IGF2 peptide. In some embodiments, thetherapeutic protein comprises at least one enzyme of the groupconsisting of alpha-galactosidase (A or B), β-galactosidase,hexosaminidase (A or B), galactosylceramidase, arylsulfatase (A or B),β-glucocerebrosidase, glucocerebrosidase, lysosomal acid lipase,lysosomal enzyme acid sphingomyelinase, formylglycine-generating enzyme,iduronidase (e.g., alpha-L), acetyl-CoA:alpha-glucosaminideN-acetyltransferase, glycosaminoglycan alpha-L-iduronohydrolase, heparanN-sulfatase, N-acetyl-α-D-glucosaminidase (NAGLU),iduronate-2-sulfatase, galactosamine-6-sulfate sulfatase,N-acetylgalactosamine-6-sulfatase, glycosaminoglycanN-acetylgalactosamine 4-sulfatase, β-glucuronidase, hyaluronidase,alpha-N-acetyl neuraminidase (sialidase), ganglioside sialidase,phosphotransferase, alpha-glucosidase, alpha-D-mannosidase,beta-D-mannosidase, aspartylglucosaminidase, alpha-L-fucosidase,battenin, palmitoyl protein thioesterases, and other Batten-relatedproteins (e.g., ceroid-lipofuscinosis neuronal protein 6), or anenzymatically active fragment thereof. In some embodiments, thetherapeutic protein comprises alpha-glucosidase, or an enzymaticallyactive fragment thereof. In some embodiments, the therapeutic protein isN-acetyl-α-D-glucosaminidase (NAGLU). In some embodiments, the nucleicacid further comprises a promoter. In some embodiments, the nucleic acidis comprised within a viral vector. In some embodiments, the viralvector comprises a retrovirus, an adenovirus, an adeno associated virus,a lentivirus, or a herpes virus. In some embodiments, the buffer orexcipient suitable for gene therapy comprises a liposome, ananoparticle, or a cell-penetrating peptide. In some embodiments, thebuffer or excipient suitable for gene therapy comprises a viral coatprotein. In some embodiments, the viral coat protein is selected fromthe group consisting of a vesicular stomatitis virus coat protein, anadenovirus coat protein, an adeno-associated virus coat protein, amurine leukemia virus coat protein, an HIV coat protein, and aninfluenza virus coat protein. In some embodiments, the genetic disorderis a lysosomal storage disorder. In some embodiments, the geneticdisorder is selected from the group consisting ofaspartylglucosaminuria, Batten disease, cystinosis, Fabry disease,Gaucher disease type I, Gaucher disease type II, Gaucher disease typeIII, Pompe disease, Tay Sachs disease, Sandhoff disease, metachomaticleukodystrophy, mucolipidosis type I, mucolipidosis type II,mucolipidosis type III, mucolipidosis type IV, Hurler disease, Hunterdisease, Sanfilippo disease type A, Sanfilippo disease type B,Sanfilippo disease type C, Sanfilippo disease type D, Morquio diseasetype A, Morquio disease type B, Maroteau-Lamy disease, Sly disease,Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pickdisease type C1, Niemann-Pick disease type C2, Schindler disease type I,Schindler disease type II, adenosine deaminase severe combinedimmunodeficiency (ADA-SCID), and chronic granulomatous disease (CGD). Insome embodiments, cells from the individual are treated ex vivo andadministered to the individual after ex vivo treatment.

In further aspects, there are provided methods of treating a geneticdisorder in an individual comprising administering a cell comprising anucleic acid encoding a fusion protein comprising a variant IGF2 peptideand a therapeutic protein. In some embodiments, the fusion proteinfurther comprises a linker. In some embodiments, the linker consists of5-20 amino acids, 5-15 amino acids, 5-10 amino acids, 8-12 amino acids,or about 7, 8, 9, 10, 11, 12 or 13 amino acids. In some embodiments, thelinker comprises an amino acid sequence of GGGGSGGGG (SEQ ID NO: 18),GGGGS (SEQ ID NO: 19), GGGSGGGGS (SEQ ID NO: 20), GGGGSGGGS (SEQ ID NO:21), GGGGSGGGGS (SEQ ID NO: 37), or GGSGSGSTS (SEQ ID NO: 33). In someembodiments, the fusion protein further comprises a signal peptide. Insome embodiments, the signal peptide comprises a binding immunoglobulinprotein (BiP) signal peptide. In some embodiments, the nucleic acidfurther comprises a cricket paralysis virus internal ribosome entrysequence (CrPV IBES). In some embodiments, the nucleic acid furthercomprises a Kozak sequence. In some embodiments, the therapeutic proteincomprises at least one enzyme of the group consisting ofalpha-galactosidase (A or B), β-galactosidase, β-hexosaminidase (A orB), galactosylceramidase, arylsulfatase (A or B), β-glucocerebrosidase,glucocerebrosidase, lysosomal acid lipase, lysosomal enzyme acidsphingomyelinase, formylglycine-generating enzyme, iduronidase (e.g.,alpha-L), acetyl-CoA:alpha-glucosaminide N-acetyltransferase,glycosaminoglycan alpha-L-iduronohydrolase, heparan N-sulfatase,N-acetyl-α-D-glucosaminidase (NAGLU), iduronate-2-sulfatase,galactosamine-6-sulfate sulfatase, N-acetylgalactosamine-6-sulfatase,glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β-glucuronidase,hyaluronidase, alpha-N-acetyl neuraminidase (sialidase), gangliosidesialidase, phosphotransferase, alpha-glucosidase, alpha-D-mannosidase,beta-D-mannosidase, aspartylglucosaminidase, alpha-L-fucosidase,battenin, palmitoyl protein thioesterases, and other Batten-relatedproteins (e.g., ceroid-lipofuscinosis neuronal protein 6), or anenzymatically active fragment thereof. In some embodiments, thetherapeutic protein comprises alpha-glucosidase, or an enzymaticallyactive fragment thereof. In some embodiments, the therapeutic protein isN-acetyl-α-D-glucosaminidase (NAGLU). In some embodiments, the nucleicacid further comprises a promoter. In some embodiments, the nucleic acidis comprised within a viral vector. In some embodiments, the viralvector comprises a retrovirus, an adenovirus, an adeno associated virus,a lentivirus, or a herpes virus. In some embodiments, the buffer orexcipient suitable for gene therapy comprises a liposome, ananoparticle, or a cell-penetrating peptide. In some embodiments, thebuffer or excipient suitable for gene therapy comprises a viral coatprotein. In some embodiments, the viral coat protein is selected fromthe group consisting of a vesicular stomatitis virus coat protein, anadenovirus coat protein, an adeno-associated virus coat protein, amurine leukemia virus coat protein, an HIV coat protein, and aninfluenza virus coat protein. In some embodiments, the genetic disorderis a lysosomal storage disorder. In some embodiments, the geneticdisorder is selected from the group consisting ofaspartylglucosaminuria, Batten disease, cystinosis, Fabry disease,Gaucher disease type I, Gaucher disease type II, Gaucher disease typeIII, Pompe disease, Tay Sachs disease, Sandhoff disease, metachomaticleukodystrophy, mucolipidosis type I, mucolipidosis type II,mucolipidosis type III, mucolipidosis type IV, Hurler disease, Hunterdisease, Sanfilippo disease type A, Sanfilippo disease type B,Sanfilippo disease type C, Sanfilippo disease type D, Morquio diseasetype A, Morquio disease type B, Maroteau-Lamy disease, Sly disease,Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pickdisease type C1, Niemann-Pick disease type C2, Schindler disease type I,Schindler disease type II, adenosine deaminase severe combinedimmunodeficiency (ADA-SCID), and chronic granulomatous disease (CGD). Insome embodiments, the cells are derived from the individual.

In additional aspects, there are provided methods of treating a geneticdisorder in an individual comprising administering a cell comprising anucleic acid encoding a fusion protein comprising a signal peptide and atherapeutic protein. In some embodiments, the signal peptide comprises abinding immunoglobulin protein (BiP) signal peptide. In someembodiments, the fusion protein further comprises a linker. In someembodiments, the linker consists of 5-20 amino acids, 5-15 amino acids,5-10 amino acids, 8-12 amino acids, or about 7, 8, 9, 10, 11, 12 or 13amino acids. In some embodiments, the linker comprises an amino acidsequence of GGGGSGGGG (SEQ ID NO: 18), GGGGS (SEQ ID NO: 19), GGGSGGGGS(SEQ ID NO: 20), GGGGSGGGS (SEQ ID NO: 21), GGGGSGGGGS (SEQ ID NO: 37),or GGSGSGSTS (SEQ ID NO: 33). In some embodiments, the fusion proteinfurther comprises a variant IGF2 peptide. In some embodiments, thenucleic acid further comprises a cricket paralysis virus internalribosome entry sequence (CrPV IBES). In some embodiments, the nucleicacid further comprises a Kozak sequence. In some embodiments, thetherapeutic protein comprises at least one enzyme of the groupconsisting of alpha-galactosidase (A or B), β-galactosidase,β-hexosaminidase (A or B), galactosylceramidase, arylsulfatase (A or B),β-glucocerebrosidase, glucocerebrosidase, lysosomal acid lipase,lysosomal enzyme acid sphingomyelinase, formylglycine-generating enzyme,iduronidase (e.g., alpha-L), acetyl-CoA:alpha-glucosaminideN-acetyltransferase, glycosaminoglycan alpha-L-iduronohydrolase, heparanN-sulfatase, N-acetyl-α-D-glucosaminidase (NAGLU),iduronate-2-sulfatase, galactosamine-6-sulfate sulfatase,N-acetylgalactosamine-6-sulfatase, glycosaminoglycanN-acetylgalactosamine 4-sulfatase, β-glucuronidase, hyaluronidase,alpha-N-acetyl neuraminidase (sialidase), ganglioside sialidase,phosphotransferase, alpha-glucosidase, alpha-D-mannosidase,beta-D-mannosidase, aspartylglucosaminidase, alpha-L-fucosidase,battenin, palmitoyl protein thioesterases, and other Batten-relatedproteins (e.g., ceroid-lipofuscinosis neuronal protein 6), or anenzymatically active fragment thereof. In some embodiments, thetherapeutic protein comprises alpha-glucosidase, or an enzymaticallyactive fragment thereof. In some embodiments, the therapeutic protein isN-acetyl-α-D-glucosaminidase (NAGLU). In some embodiments, the nucleicacid further comprises a promoter. In some embodiments, the nucleic acidis comprised within a viral vector. In some embodiments, the viralvector comprises a retrovirus, an adenovirus, an adeno associated virus,a lentivirus, or a herpes virus. In some embodiments, the buffer orexcipient suitable for gene therapy comprises a liposome, ananoparticle, or a cell-penetrating peptide. In some embodiments, thebuffer or excipient suitable for gene therapy comprises a viral coatprotein. In some embodiments, the viral coat protein is selected fromthe group consisting of a vesicular stomatitis virus coat protein, anadenovirus coat protein, an adeno-associated virus coat protein, amurine leukemia virus coat protein, an HIV coat protein, and aninfluenza virus coat protein. In some embodiments, the genetic disorderis a lysosomal storage disorder. In some embodiments, the geneticdisorder is selected from the group consisting ofaspartylglucosaminuria, Batten disease, cystinosis, Fabry disease,Gaucher disease type I, Gaucher disease type II, Gaucher disease typeIII, Pompe disease, Tay Sachs disease, Sandhoff disease, metachomaticleukodystrophy, mucolipidosis type I, mucolipidosis type II,mucolipidosis type III, mucolipidosis type IV, Hurler disease, Hunterdisease, Sanfilippo disease type A, Sanfilippo disease type B,Sanfilippo disease type C, Sanfilippo disease type D, Morquio diseasetype A, Morquio disease type B, Maroteau-Lamy disease, Sly disease,Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pickdisease type C1, Niemann-Pick disease type C2, Schindler disease type I,Schindler disease type II, adenosine deaminase severe combinedimmunodeficiency (ADA-SCID), and chronic granulomatous disease (CGD). Insome embodiments, the cells are derived from the individual.

In additional aspects, there are provided methods of treating a geneticdisorder in an individual comprising administering a cell comprising anucleic acid encoding a fusion protein comprising a therapeutic protein,wherein the nucleic acid further comprises a cricket paralysis virusinternal ribosome entry sequence (CrPV IBES). In some embodiments, thenucleic acid further comprises a Kozak sequence. In some embodiments,the fusion protein further comprises a linker. In some embodiments, thelinker consists of 5-20 amino acids, 5-15 amino acids, 5-10 amino acids,8-12 amino acids, or about 7, 8, 9, 10, 11, 12 or 13 amino acids. Insome embodiments, the linker comprises an amino acid sequence ofGGGGSGGGG (SEQ ID NO: 18), GGGGS (SEQ ID NO: 19), GGGSGGGGS (SEQ ID NO:20), GGGGSGGGS (SEQ ID NO: 21), GGGGSGGGGS (SEQ ID NO: 37), or GGSGSGSTS(SEQ ID NO: 33). In some embodiments, the fusion protein furthercomprises a signal peptide. In some embodiments, the signal peptidecomprises a binding immunoglobulin protein (BiP) signal peptide. In someembodiments, the fusion protein further comprises a variant IGF2peptide. In some embodiments, the therapeutic protein comprises at leastone enzyme of the group consisting of alpha-galactosidase (A or B),β-galactosidase, β-hexosaminidase (A or B), galactosylceramidase,arylsulfatase (A or B), β-glucocerebrosidase, glucocerebrosidase,lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase,formylglycine-generating enzyme, iduronidase (e.g., alpha-L),acetyl-CoA:alpha-glucosaminide N-acetyltransferase, glycosaminoglycanalpha-L-iduronohydrolase, heparan N-sulfatase,N-acetyl-α-D-glucosaminidase (NAGLU), iduronate-2-sulfatase,galactosamine-6-sulfate sulfatase, N-acetylgalactosamine-6-sulfatase,glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β-glucuronidase,hyaluronidase, alpha-N-acetyl neuraminidase (sialidase), gangliosidesialidase, phosphotransferase, alpha-glucosidase, alpha-D-mannosidase,beta-D-mannosidase, aspartylglucosaminidase, alpha-L-fucosidase,battenin, palmitoyl protein thioesterases, and other Batten-relatedproteins (e.g., ceroid-lipofuscinosis neuronal protein 6), or anenzymatically active fragment thereof. In some embodiments, thetherapeutic protein comprises alpha-glucosidase, or an enzymaticallyactive fragment thereof. In some embodiments, the therapeutic protein isN-acetyl-α-D-glucosaminidase (NAGLU). In some embodiments, the nucleicacid further comprises a promoter. In some embodiments, the nucleic acidis comprised within a viral vector. In some embodiments, the viralvector comprises a retrovirus, an adenovirus, an adeno associated virus,a lentivirus, or a herpes virus. In some embodiments, the buffer orexcipient suitable for gene therapy comprises a liposome, ananoparticle, or a cell-penetrating peptide. In some embodiments, thebuffer or excipient suitable for gene therapy comprises a viral coatprotein. In some embodiments, the viral coat protein is selected fromthe group consisting of a vesicular stomatitis virus coat protein, anadenovirus coat protein, an adeno-associated virus coat protein, amurine leukemia virus coat protein, an HIV coat protein, and aninfluenza virus coat protein. In some embodiments, the genetic disorderis a lysosomal storage disorder. In some embodiments, the geneticdisorder is selected from the group consisting ofaspartylglucosaminuria, Batten disease, cystinosis, Fabry disease,Gaucher disease type I, Gaucher disease type II, Gaucher disease typeIII, Pompe disease, Tay Sachs disease, Sandhoff disease, metachomaticleukodystrophy, mucolipidosis type I, mucolipidosis type II,mucolipidosis type III, mucolipidosis type IV, Hurler disease, Hunterdisease, Sanfilippo disease type A, Sanfilippo disease type B,Sanfilippo disease type C, Sanfilippo disease type D, Morquio diseasetype A, Morquio disease type B, Maroteau-Lamy disease, Sly disease,Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pickdisease type C1, Niemann-Pick disease type C2, Schindler disease type I,Schindler disease type II, adenosine deaminase severe combinedimmunodeficiency (ADA-SCID), and chronic granulomatous disease (CGD). Insome embodiments, the cells are derived from the individual.

In further aspects, there are provided methods of treating a geneticdisorder in an individual comprising administering a cell comprising anucleic acid encoding a fusion protein comprising a therapeutic protein,wherein the nucleic acid further comprises a Kozak sequence. In someembodiments, the nucleic acid further comprises a cricket paralysisvirus internal ribosome entry sequence (CrPV IBES). In some embodiments,the fusion protein further comprises a linker. In some embodiments, thelinker consists of 5-20 amino acids, 5-15 amino acids, 5-10 amino acids,8-12 amino acids, or about 7, 8, 9, 10, 11, 12 or 13 amino acids. Insome embodiments, the linker comprises an amino acid sequence ofGGGGSGGGG (SEQ ID NO: 18), GGGGS (SEQ ID NO: 19), GGGSGGGGS (SEQ ID NO:20), GGGGSGGGS (SEQ ID NO: 21), GGGGSGGGGS (SEQ ID NO: 37), or GGSGSGSTS(SEQ ID NO: 33). In some embodiments, the fusion protein furthercomprises a signal peptide. In some embodiments, the signal peptidecomprises a binding immunoglobulin protein (BiP) signal peptide. In someembodiments, the fusion protein further comprises a variant IGF2peptide. In some embodiments, the therapeutic protein comprises at leastone enzyme of the group consisting of alpha-galactosidase (A or B),β-galactosidase, β-hexosaminidase (A or B), galactosylceramidase,arylsulfatase (A or B), β-glucocerebrosidase, glucocerebrosidase,lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase,formylglycine-generating enzyme, iduronidase (e.g., alpha-L),acetyl-CoA:alpha-glucosaminide N-acetyltransferase, glycosaminoglycanalpha-L-iduronohydrolase, heparan N-sulfatase,N-acetyl-α-D-glucosaminidase (NAGLU), iduronate-2-sulfatase,galactosamine-6-sulfate sulfatase, N-acetylgalactosamine-6-sulfatase,glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β-glucuronidase,hyaluronidase, alpha-N-acetyl neuraminidase (sialidase), gangliosidesialidase, phosphotransferase, alpha-glucosidase, alpha-D-mannosidase,beta-D-mannosidase, aspartylglucosaminidase, alpha-L-fucosidase,battenin, palmitoyl protein thioesterases, and other Batten-relatedproteins (e.g., ceroid-lipofuscinosis neuronal protein 6), or anenzymatically active fragment thereof. In some embodiments, thetherapeutic protein comprises alpha-glucosidase, or an enzymaticallyactive fragment thereof. In some embodiments, the therapeutic protein isN-acetyl-α-D-glucosaminidase (NAGLU). In some embodiments, the nucleicacid further comprises a promoter. In some embodiments, the nucleic acidis comprised within a viral vector. In some embodiments, the viralvector comprises a retrovirus, an adenovirus, an adeno associated virus,a lentivirus, or a herpes virus. In some embodiments, the buffer orexcipient suitable for gene therapy. In some embodiments, the buffer orexcipient suitable for gene therapy comprises a viral coat protein. Insome embodiments, the viral coat protein is selected from the groupconsisting of a vesicular stomatitis virus coat protein, an adenoviruscoat protein, an adeno-associated virus coat protein, a murine leukemiavirus coat protein, an HIV coat protein, and an influenza virus coatprotein. In some embodiments, the genetic disorder is a lysosomalstorage disorder. In some embodiments, the genetic disorder is selectedfrom the group consisting of aspartylglucosaminuria, Batten disease,cystinosis, Fabry disease, Gaucher disease type I, Gaucher disease typeII, Gaucher disease type III, Pompe disease, Tay Sachs disease, Sandhoffdisease, metachomatic leukodystrophy, mucolipidosis type I,mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV,Hurler disease, Hunter disease, Sanfilippo disease type A, Sanfilippodisease type B, Sanfilippo disease type C, Sanfilippo disease type D,Morquio disease type A, Morquio disease type B, Maroteau-Lamy disease,Sly disease, Niemann-Pick disease type A, Niemann-Pick disease type B,Niemann-Pick disease type C1, Niemann-Pick disease type C2, Schindlerdisease type I, Schindler disease type II, adenosine deaminase severecombined immunodeficiency (ADA-SCID), and chronic granulomatous disease(CGD). In some embodiments, the cells are derived from the individual.

Further provided herein is a fusion protein comprising a native signalpeptide, an ER proteolytic cleavage domain, a variant IGF2 peptide, andan alpha-glucosidase lacking its native signal peptide, wherein thefusion protein is encoded by a nucleic acid comprising a Kozak sequence.

Additionally provided herein is a fusion protein comprising a bindingimmunoglobulin protein (BiP) signal peptide, a variant IGF2 peptide, andan alpha-glucosidase, wherein the fusion protein is encoded by a nucleicacid comprising a Kozak sequence.

Additionally provided herein is a fusion protein comprising a bindingimmunoglobulin protein (BiP) signal peptide, a variant IGF2 peptide, andan alpha-glucosidase lacking its native signal peptide, wherein thefusion protein is encoded by a nucleic acid comprising a cricketparalysis virus internal ribosome entry sequence (CrPV IRES).

Additionally provided herein is a nucleic acid encoding a fusion proteincomprising a native signal peptide, an ER proteolytic cleavage domain, avariant IGF2 peptide, and an alpha-glucosidase lacking its native signalpeptide.

Additionally provided herein is a nucleic acid encoding a fusion proteincomprising a binding immunoglobulin protein (BiP) signal peptide, avariant IGF2 peptide and an alpha-glucosidase lacking its native signalpeptide, wherein the nucleic acid further comprises a Kozak sequence.

Additionally provided herein is a nucleic acid encoding a fusion proteincomprising a variant IGF2 peptide and an alpha-glucosidase, wherein thenucleic acid further comprises a cricket paralysis virus internalribosome entry sequence (CrPV IRES).

Additionally provided herein is a composition comprising (a) a nucleicacid encoding a fusion protein comprising a native signal peptide, an ERproteolytic cleavage domain, a variant IGF2 peptide, and analpha-glucosidase lacking its native signal peptide; and (b) a buffer orexcipient suitable for gene therapy.

Additionally provided herein is a composition comprising (a) a nucleicacid encoding a fusion protein comprising a binding immunoglobulinprotein (BiP) signal peptide, a variant IGF2 peptide and analpha-glucosidase lacking its native signal peptide, wherein the nucleicacid further comprises a Kozak sequence; and (b) a buffer or excipientsuitable for gene therapy.

Additionally provided herein is a composition comprising (a) a nucleicacid encoding a fusion protein comprising a variant IGF2 peptide and analpha-glucosidase, wherein the nucleic acid further comprises a cricketparalysis virus internal ribosome entry sequence (CrPV IRES); and (b) abuffer or excipient suitable for gene therapy.

Additionally provided herein is a method of treating Pompe disease in anindividual comprising administering a composition comprising (a) anucleic acid encoding a fusion protein comprising a native signalpeptide, an ER proteolytic cleavage domain, a variant IGF2 peptide, andan alpha-glucosidase lacking its native signal peptide; and (b) a bufferor excipient suitable for gene therapy.

Additionally provided herein is a method of treating Pompe disease in anindividual comprising administering a composition comprising (a) anucleic acid encoding a fusion protein comprising a bindingimmunoglobulin protein (BiP) signal peptide, a variant IGF2 peptide andan alpha-glucosidase lacking its native signal peptide, wherein thenucleic acid further comprises a Kozak sequence; and (b) a buffer orexcipient suitable for gene therapy.

Additionally provided herein is a method of treating Pompe disease in anindividual comprising administering a composition comprising (a) anucleic acid encoding a fusion protein comprising a variant IGF2 peptideand an alpha-glucosidase, wherein the nucleic acid further comprises acricket paralysis virus internal ribosome entry sequence (CrPV IRES);and (b) a buffer or excipient suitable for gene therapy.

Additionally provided herein is a method of treating Pompe disease in anindividual comprising administering a cell comprising a nucleic acidencoding a fusion protein comprising a native signal peptide, an ERproteolytic cleavage domain, a variant IGF2 peptide, and analpha-glucosidase lacking its native signal peptide.

Additionally provided herein is a method of treating Pompe disease in anindividual comprising administering a cell comprising a nucleic acidencoding a fusion protein comprising a binding immunoglobulin protein(BiP) signal peptide, a variant IGF2 peptide and an alpha-glucosidase,or an enzymatically active fragment thereof, wherein the nucleic acidfurther comprises a Kozak sequence.

Additionally provided herein is a method of treating Pompe disease in anindividual comprising administering a cell comprising a nucleic acidencoding a fusion protein comprising a variant IGF2 peptide and analpha-glucosidase, wherein the nucleic acid further comprises a cricketparalysis virus internal ribosome entry sequence (CrPV IRES).

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent application file contains at least one drawing executed incolor. Copies of this patent application with color drawing(s) will beprovided by the Office upon request and payment of the necessary fee. Anunderstanding of the features and advantages of the present disclosurewill be obtained by reference to the following detailed description thatsets forth illustrative embodiments, in which the principles of thedisclosure are utilized, and the accompanying drawings of which:

FIG. 1 shows GAA activity of alglucosidase-alfa rhGAA with and withoutM6P. FIG. 1 shows the proportion of commercial ERT that is able to bindto the CI-MPR. The first peak is the rhGAA that lack any M6P containingglycans and thus unable to be taken up and delivered to the lysosome.The second peak is the fraction that contains at least onephosphorylated glycan and has the potential to be taken up by the celland delivered to the lysosome for hydrolysis of glycogen.

FIG. 2 shows structure of the CI-MPR including the different bindingdomains for the IGF2 and for mono- and bis-phosphorylatedoligosaccharides.

FIG. 3 shows the sequence and structure of the mature, human IGF2peptide. Site specific amino acid substitutions are proposed toinfluence binding of other receptors.

FIG. 4 shows binding of the wild-type IGF2 (wtIGF2) peptide to CI-MPR asmeasured by surface plasmon resonance

FIG. 5 shows binding of the variant IGF2 (vIGF2) peptide binding toCI-MPR as measured by surface plasmon resonance.

FIG. 6 shows benefit of adding vIGF2 to aluglucisidase alfa to increasethe binding to the IGF2/CI-MPR.

FIG. 7 shows the benefit of adding a vIGF2 to recombinant humanN-acetyl-α-D-glucosaminidase (rhNAGLU) to increase the binding to theIGF2/CI-MPR.

FIG. 8 shows binding of wildtype human IGF2 to insulin receptor.

FIG. 9 shows no detectable binding of vIGF2 to insulin receptor.

FIG. 10 shows binding of wildtype IGF2 to insulin-like growth factor 1receptor.

FIG. 11 shows decreased binding of vIGF2 peptide to insulin-like growthfactor 1 receptor, as compared to wildtype IGF2.

FIG. 12 shows two examples of gene therapy expression cassettes encodingNatural hGAA and Engineered hGAA. Natural hGAA has poor phosphorylationleading to poor CIMPR binding and cellular uptake. Engineered hGAA haselement added for improved CIMPR binding (vIGF2), a 2GS linker thatreduces steric hinderance of the vIGF2-GAA protein with CIMPR, and a BiPsignal peptide to improve secretion.

FIG. 13 shows a Western blot of PPT1 from cells expressing recombinanthuman PPT1 (PPT1-1), recombinant human PPT1 having a vIGF2 targetingdomain (PPT1-2) and recombinant human PPT1 having a vIGF2 targetingdomain and a BiP signal sequence (PPT1-29).

FIG. 14 shows binding of PPT1 constructs to CI-MPR.

FIG. 15 shows GAA activity in conditioned media of CHO cells expressingengineered or natural hGAA.

FIG. 16 shows the study design of a 4 week mouse study of gene therapyin a GAA knockout mouse.

FIG. 17 shows GAA plasma activity in untreated wild type (“Normal”) miceor GAA knockout mice treated with gene therapy vectors or vehicle asindicated.

FIG. 18 shows GAA levels measured in untreated wild type (“Normal”) miceor GAA knockout mice treated with gene therapy vectors or vehicle asindicated.

FIG. 19 shows cell surface receptor binding of rhGAA from plasma samplesobtained from treated mice as indicated.

FIG. 20 shows GAA activity, and quad glycogen histopathology score fortibialis antierior of untreated wild type (“Normal”) mice or GAAknockout mice treated with gene therapy vectors or vehicle as indicated.

FIG. 21 shows glycogen PAS of tibialis antierior from untreated wildtype mice or GAA knockout mice treated with gene therapy vectors orvehicle as indicated.

FIG. 22 shows hGAA immunohistochemistry of tibialis antierior fromuntreated wild type mice or GAA knockout mice treated with gene therapyvectors or vehicle as indicated.

FIG. 23 shows brain GAA activity, brain glycogen, and spinal cordglycogen histopathology scoring for brain and spinal cord from untreatedwild type (“Normal”) mice or GAA knockout mice treated with gene therapyvectors or vehicle as indicated.

FIG. 24 shows glycogen PAS of brain from untreated wild type mice or GAAknockout mice treated with gene therapy vectors or vehicle as indicated.

FIG. 25 shows hGAA immunohistochemistry of brainstem and choroid plexusfrom untreated wild type (“Normal”) mice or GAA knockout mice treatedwith gene therapy vectors or vehicle as indicated.

FIG. 26 shows glycogen PAS of spinal cord from untreated wild type miceor GAA knockout mice treated with gene therapy vectors or vehicle asindicated.

FIG. 27 shows hGAA immunohistochemistry of spinal cord from untreatedwild type mice or GAA knockout mice treated with gene therapy vectors orvehicle as indicated.

FIG. 28 shows quadriceps GAA activity and glycogen histopathologyscoring from untreated wild type (“Normal”) mice or GAA knockout micetreated with gene therapy vectors or vehicle as indicated.

FIG. 29 shows glycogen luxol/PAS for quadriceps from untreated wild typemice or GAA knockout mice treated with gene therapy vectors or vehicleas indicated.

FIG. 30 shows hGAA immunohistochemistry of quadriceps from untreatedwild type mice or GAA knockout mice treated with gene therapy vectors orvehicle as indicated.

FIG. 31 shows triceps GAA activity and histopathology scoring foruntreated wild type (“Normal”) mice or GAA knockout mice treated withgene therapy vectors or vehicle as indicated.

FIG. 32 shows glycogen luxol/PAS of triceps from untreated wild typemice or GAA knockout mice treated with gene therapy vectors or vehicleas indicated.

FIG. 33 shows hGAA immunohistochemistry of triceps from untreated wildtype mice or GAA knockout mice treated with gene therapy vectors orvehicle as indicated.

DETAILED DESCRIPTION

Gene therapy for single gene genetic disorders presents a potentialone-time treatment for diseases and disorders, some of which havedevastating symptoms that can appear early in life and sometimes lead tolife-long disability. Neurologic genetic disorders, such as lysosomalstorage disorders, are often treated with enzyme replacement therapieswhich administer to the patient a therapeutic protein that is an activeform of the protein that is defective or deficient in the disease ordisorder state. However, there are challenges for current therapies,including frequent treatments, development of an immune response to thetherapeutic protein, and difficulty targeting the therapeutic protein tothe affected tissue, cell, or subcellular compartment. Gene therapyoffers advantages including a reduced number of treatments and longlasting efficacy.

Provided herein are components for gene therapy vectors that offerimprovements to gene therapy, such as providing more therapeutic proteinwhere it is needed, thus improving treatment efficacy. Such challengesare addressed herein by improving expression and cellular uptake ordelivery and intracellular or subcellular targeting of therapeuticproteins. Specific tools or components provided herein include but arenot limited to signal peptides (e.g., binding immunoglobulin protein(BiP) and Gaussia signal peptides) for increasing secretion and peptidesthat increase endocytosis of the therapeutic protein (e.g., peptidesthat bind to the CI-MPR with high affinity for increasing cellularuptake and lysosomal delivery). Such peptides are fused to therapeuticproteins encoded by gene therapy vectors. In some embodiments, thepeptides are IGF2 (Insulin Like growth factor 2) peptides or variantsthereof. Gene therapy vectors provided herein are contemplated tocomprise, in some embodiments, a nucleic acid encoding a therapeuticprotein fused to a peptide that bind to the CI-MPR with high affinityfor optimizing efficacy of gene therapy.

Gene therapy constructs for enzyme replacement gene therapy weredesigned. A translation initiation sequence, including, but not limitedto a Kozak sequence or an IRES sequence, such as CrPV IRES, located atthe 5′ end of the construct, followed by a nucleic acid encoding asignal peptide selected from one or more of a GAA signal peptide, anucleic acid encoding an anti-trypsin inhibitor, and a nucleic acidencoding BiP sequence. These are followed by a nucleic acid encoding acell targeting domain which can be a vIGF-2, a HIRMab, or a TfRMab orother cell targeting peptide or protein. The gene therapy constructfurther comprises a nucleic acid encoding a linker and a nucleic acidencomding a corrective enzyme or enzymatically active fragment thereof,wherein the linker connects the cell targeting domain to the correctiveenzyme, or enzymatically active fragment thereof. Suitable correctiveenzymes include but are not limited to alpha-glucosidase (GAA),alpha-galactosidase (GLA), iduronidase (IDUA), iduroniate-2-sulfatase(IDS), PPT1, or enzymatically active fragments thereof, and otherenzymes found deficient in an individual.

Intracellular Targeting of Therapeutic Proteins

N-linked carbohydrates of most lysosomal proteins are modified tocontain a specialized carbohydrate structure called mannose 6-phosphate(M6P). M6P is the biological signal that enables transport of lysosomalproteins to lysosomes via membrane-bound M6P receptors. Enzymereplacement therapies for lysosomal storage disorders utilize M6Preceptors for uptake and delivery of therapeutic proteins to lysosomes.Certain therapeutics do not utilize M6P receptors including Cerezyme®and other versions of recombinant human GCase, utilize the mannosereceptor that is able to bind terminal mannose on protein glycans anddeliver to the lysosome. A problem facing certain enzyme replacementtherapeutics is there are low amounts of M6P present on the enzymetherapeutic which necessitate higher doses to reach therapeuticefficacy. This leads to substantially longer infusion times, higherprobability of developing immune responses to the therapeutic, andhigher drug demand, requiring increased protein manufacturing resultingin increased costs.

The CI-MPR captures M6P-containing lysosomal enzymes from circulation.The receptor has distinct binding domains for M6P and insulin-likegrowth factor (domains 1-3 and 7-9, see FIG. 2 ) and therefore is alsoknown as the IGF2/Mannose-6-phosphate receptor or IGF2/CI-MPR. Thisreceptor can be utilized for targeting M6P- or IGF2- or IGF2variant-containing enzyme replacement therapeutics. Binding affinity ofthis receptor for these ligands including insulin-like growth factor isprovided in Table 1. Notably, IGF2 peptide has a higher binding affinityfor CI-MPR than mono- or bis-phosphorylated oligosaccharides.

TABLE 1 Ligands for CI-MPR Ligand Binding Affinity (Apparent Kd; nM)IGF2 0.03-0.2 [Leu27]IGF2 0.05 Bis-M6P 2 Beta-galactosidase 20Pentamannose-M6P 6,000 Free M6P 7,000

Therapeutic Fusion Proteins for Gene Therapy

Therapeutic fusion proteins produced from gene therapy vectors areprovided herein. In some embodiments the fusion protein is secreted bycells transduced with the gene therapy vector encoding the fusionprotein. In some embodiments, the transduced cells are within a tissueor organ (e.g., liver). Once secreted from a cell, the fusion protein istransported through a patient's vascular system and reaches the tissueof interest. In some embodiments, the therapeutic fusion protein isengineered to have improved secretion. In some embodiments, the fusionprotein comprises a signal peptide for improving the secretion level ascompared to the corresponding therapeutic protein or a fusion proteincomprising the therapeutic protein but lacking a signal peptide.

The provided gene therapy vectors are, in some embodiments, engineeredto address issues with gene therapy with regard to delivery of thetherapeutic protein. For example, in some instances gene therapy may notachieve the intended treatment by merely generating a sufficient amountof a therapeutic protein in the body of the patient if an insufficientamount of the therapeutic protein is delivered into the cells in need ofthe therapeutic protein, due to, for example, physical and/or biologicalbarriers that impede distribution of the therapeutic protein to the sitewhere needed. As such, even if a gene therapy is capable of floodingblood or a tissue, to a point of saturation, with a high concentrationof a therapeutic protein, the gene therapy may not be sufficientlytherapeutic. Additionally, non-productive clearance pathways may removethe vast majority of the therapeutic protein. Even if the therapeuticprotein is transported out of the vasculature to the interstitial spacewithin the tissue (e.g., muscle fibers), adequate therapeutic effectsare not assured. For effective treatment of lysosomal storage disorders,a therapeutically effective amount of the therapeutic protein mustundergo cellular endocytosis and lysosomal delivery to result in ameaningful efficacy. The present disclosure addresses these issues byproviding gene therapy vectors encoding fusion proteins comprising apeptide that enables endocytosis of the therapeutic protein into atarget cell for treatment resulting in efficacious treatment. In someembodiments, the peptide that enables endocytosis is a peptide thatbinds the CI-MPR. In some embodiments, the peptide that binds the CI-MPRis a vIGF2 peptide.

Provided herein are gene therapy vectors encoding fusion proteinscomprising a peptide that enables endocytosis the therapeutic proteininto a target cell for treatment. In some embodiments, the gene therapyvectors encode fusion proteins comprising a therapeutic protein and apeptide that binds the CI-MPR. Such fusion proteins when expressed froma gene therapy vector target therapeutic proteins, such as enzymereplacement therapeutics, to the cells where they are needed, increasedelivery into or cellular uptake by such cells and target thetherapeutic protein to a subcellular location (e.g., a lysosome). Insome embodiments, the peptide is an IGF2 peptide or variant thereof,which can target a therapeutic protein to the lysosome. Fusion proteinsherein also, in some embodiments, further comprise a signal peptide thatincreases secretion, such as a BiP signal peptide or a Gaussia signalpeptide. In some embodiments, fusion proteins comprise a linkersequence. In some embodiments, nucleic acids encoding fusion proteinsherein, comprise internal ribosomal entry sequences.

Therapeutic proteins for gene therapy comprising a vIGF2 peptide areprovided herein. Exemplary proteins are provided in Table 2 below.

TABLE 2 SEQ ID  Amino Acid Sequences NO NaturalMGVRHPPCSHRLLAVCALVSLATAALLGHILLHDFLLVPRELSGSSPVLEE 22 hGAATHPAHQQGASRPGPRDAQAHPGRPRAVPTQCDVPPNSRFDCAPDKAITQEQCEARGCCYIPAKQGLQGAQMGQPWCFFPPSYPSYKLENLSSSEMGYTATLTRTTPTFFPKDILTLRLDVMMETENRLHFTIKDPANRRYEVPLETPHVHSRAPSPLYSVEFSEEPFGVIVRRQLDGRVLLNTTVAPLFFADQFLQLSTSLPSQYITGLAEHLSPLMLSTSWTRITLWNRDLAPTPGANLYGSHPFYLALEDGGSAHGVFLLNSNAMDVVLQPSPALSWRSTGGILDVYIFLGPEPKSVVQQYLDVVGYPFMPPYWGLGFHLCRWGYSSTAITRQVVENMTRAHFPLDVQWNDLDYMDSRRDFTFNKDGFRDFPAMVQELHQGGRRYMMIVDPAISSSGPAGSYRPYDEGLRRGVFITNETGQPLIGKVWPGSTAFPDFTNPTALAWWEDMVAEFHDQVPFDGMWIDMNEPSNFIRGSEDGCPNNELENPPYVPGVVGGTLQAATICASSHQFLSTHYNLHNLYGLTEAIASHRALVKARGTRPFVISRSTFAGHGRYAGHWTGDVWSSWEQLASSVPEILQFNLLGVPLVGADVCGFLGNTSEELCVRWTQLGAFYPFMRNHNSLLSLPQEPYSFSEPAQQAMRKALTLRYALLPHLYTLFHQAHVAGETVARPLFLEFPKDSSTWTVDHQLLWGEALLITPVLQAGKAEVTGYFPLGTWYDLQTVPVEALGSLPPPPAAPREPAIHSEGQWVTLPAPLDTINVHLRAGYIIPLQGPGLTTTESRQQPMALAVALTKGGEARGELFWDDGESLEVLERGAYTQVIFLARNNTIVNELVRVTSEGAGLQLQKVTVLGVATAPQQVLSNGVPVSNFTYSPDTKVLDICVSLLMGEQFLVSWC EngineeredMKLSLVAAMLLLLSAARASRTLCGGELVDTLQFVCGDRGFLFSRPASRVS 23 hGAA (BiP-RRSRGIVEECCFRSCDLALLETYCATPARSEGGGGSGGGGSRPGPRDAQAH vIGF2-GAA)PGRPRAVPTQCDVPPNSRFDCAPDKAITQEQCEARGCCYIPAKQGLQGAQMGQPWCFFPPSYPSYKLENLSSSEMGYTATLTRTTPTFFPKDILTLRLDVMMETENRLHFTIKDPANRRYEVPLETPHVHSRAPSPLYSVEFSEEPFGVIVRRQLDGRVLLNTTVAPLFFADQFLQLSTSLPSQYITGLAEHLSPLMLSTSWTRITLWNRDLAPTPGANLYGSHPFYLALEDGGSAHGVFLLNSNAMDVVLQPSPALSWRSTGGILDVYIFLGPEPKSVVQQYLDVVGYPFMPPYWGLGFHLCRWGYSSTAITRQVVENMTRAHFPLDVQWNDLDYMDSRRDFTFNKDGFRDFPAMVQELHQGGRRYMMIVDPAISSSGPAGSYRPYDEGLRRGVFITNETGQPLIGKVWPGSTAFPDFTNPTALAWWEDMVAEFHDQVPFDGMWIDMNEPSNFIRGSEDGCPNNELENPPYVPGVVGGTLQAATICASSHQFLSTHYNLHNLYGLTEAIASHRALVKARGTRPFVISRSTFAGHGRYAGHWTGDVWSSWEQLASSVPEILQFNLLGVPLVGADVCGFLGNTSEELCVRWTQLGAFYPFMRNHNSLLSLPQEPYSFSEPAQQAMRKALTLRYALLPHLYTLFHQAHVAGETVARPLFLEFPKDSSTWTVDHQLLWGEALLITPVLQAGKAEVTGYFPLGTWYDLQTVPVEALGSLPPPPAAPREPAIHSEGQWVTLPAPLDTINVHLRAGYIIPLQGPGLTTTESRQQPMALAVALTKGGEARGELFWDDGESLEVLERGAYTQVIFLARNNTIVNELVRVTSEGAGLQLQKVTVLGVATAPQQVLSNGVPVSNFTYSPDTKVLDICVSLLMGEQFLVSWC hGAA Δ1-SRPGPRDAQAHPGRPRAVPTQCDVPPNSRFDCAPDKAITQEQCEARGCCYI 46 60PAKQGLQGAQMGQPWCFFPPSYPSYKLENLSSSEMGYTATLTRTTPTFFPKDILTLRLDVMMETENRLHFTIKDPANRRYEVPLETPHVHSRAPSPLYSVEFSEEPFGVIVRRQLDGRVLLNTTVAPLFFADQFLQLSTSLPSQYITGLAEHLSPLMLSTSWTRITLWNRDLAPTPGANLYGSHPFYLALEDGGSAHGVFLLNSNAMDVVLQPSPALSWRSTGGILDVYIFLGPEPKSVVQQYLDVVGYPFMPPYWGLGFHLCRWGYSSTAITRQVVENMTRAHFPLDVQWNDLDYMDSRRDFTFNKDGFRDFPAMVQELHQGGRRYMMIVDPAISSSGPAGSYRPYDEGLRRGVFITNETGQPLIGKVWPGSTAFPDFTNPTALAWWEDMVAEFHDQVPFDGMWIDMNEPSNFIRGSEDGCPNNELENPPYVPGVVGGTLQAATICASSHQFLSTHYNLHNLYGLTEAIASHRALVKARGTRPFVISRSTFAGHGRYAGHWTGDVWSSWEQLASSVPEILQFNLLGVPLVGADVCGFLGNTSEELCVRWTQLGAFYPFMRNHNSLLSLPQEPYSFSEPAQQAMRKALTLRYALLPHLYTLFHQAHVAGETVARPLFLEFPKDSSTWTVDHQLLWGEALLITPVLQAGKAEVTGYFPLGTWYDLQTVPVEALGSLPPPPAAPREPAIHSEGQWVTLPAPLDTINVHLRAGYIIPLQGPGLTTTESRQQPMALAVALTKGGEARGELFWDDGESLEVLERGAYTQVIFLARNNTIVNELVRVTSEGAGLQLQKVTVLGVATAPQQVLSNGVPVSNFTYSPDTKVLDICVSLLMGEQFLVSWC wt-PPT1MASPGCLWLLAVALLPWTCASRALQHLDPPAPLPLVIWHGMGDSCCNPL 24SMGAIKKMVEKKIPGIYVLSLEIGKTLMEDVENSFFLNVNSQVTTVCQALAKDPKLQQGYNAMGFSQGGQFLRAVAQRCPSPPMINLISVGGQHQGVFGLPRCPGESSHICDFIRKTLNAGAYSKVVQERLVQAEYWHDPIKEDVYRNHSIFLADINQERGINESYKKNLMALKKFVMVKFLNDSIVDPVDSEWFGFYRSGQAKETIPLQETSLYTQDRLGLKEMDNAGQLVFLATEGDHLQLSEEWFYAHII PFLG PPT1-2MASPGCLWLLAVALLPWTCASRALQHLSRTLCGGELVDTLQFVCGDRGF 25 (vIGF2-LFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPARSEGGGGSGGGGS PPT1)RPRAVPTQDPPAPLPLVIWHGMGDSCCNPLSMGAIKKMVEKKIPGIYVLSLEIGKTLMEDVENSFFLNVNSQVTTVCQALAKDPKLQQGYNAMGFSQGGQFLRAVAQRCPSPPMINLISVGGQHQGVFGLPRCPGESSHICDFIRKTLNAGAYSKVVQERLVQAEYWHDPIKEDVYRNHSIFLADINQERGINESYKKNLMALKKFVMVKFLNDSIVDPVDSEWFGFYRSGQAKETIPLQETSLYTQDRLGLKEMDNAGQLVFLATEGDHLQLSEEWFYAHIIPFLG PPT1-29MKLSLVAAMLLLLWVALLLLSAARAAASRTLCGGELVDTLQFVCGDRGF 26 (BiP2aa-LFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPARSEGGGGSGGGGS vIGF2-RPRAVPTQDPPAPLPLVIWHGMGDSCCNPLSMGAIKKMVEKKIPGIYVLSL PPT1)EIGKTLMEDVENSFFLNVNSQVTTVCQALAKDPKLQQGYNAMGFSQGGQFLRAVAQRCPSPPMINLISVGGQHQGVFGLPRCPGESSHICDFIRKTLNAGAYSKVVQERLVQAEYWHDPIKEDVYRNHSIFLADINQERGINESYKKNLMALKKFVMVKFLNDSIVDPVDSEWFGFYRSGQAKETIPLQETSLYTQDRLGLKEMDNAGQLVFLATEGDHLQLSEEWFYAHIIPFLG

Components of fusion proteins provided herein are further describedbelow.

Peptides that Bind CI-MPR (e.g., vIGF2 Peptides)

Provided herein are peptides that bind CI-MPR. Fusion proteinscomprising such peptides and a therapeutic protein, when expressed froma gene therapy vector, target the therapeutic protein to the cells whereit is needed, increase cellular uptake by such cells and target thetherapeutic protein to a subcellular location (e.g., a lysosome). Insome embodiments, the peptide is fused to the N-terminus of thetherapeutic peptide. In some embodiments, the peptide is fused to theC-terminus of the therapeutic protein. In some embodiments, the peptideis a vIGF2 peptide. Some vIGF2 peptides maintain high affinity bindingto CI-MPR while their affinity for IGF1 receptor, insulin receptor, andIGF binding proteins (IGFBP) is decreased or eliminated. Thus, somevariant IGF2 peptides are substantially more selective and have reducedsafety risks compared to wt IGF2. vIGF2 peptides herein include thosehaving the amino acid sequence of SEQ ID NO: 31. Variant IGF2 peptidesfurther include those with variant amino acids at positions 6, 26, 27,43, 48, 49, 50, 54, 55, or 65 compared to wt IGF2 (SEQ ID NO: 1). Insome embodiments, the vIGF2 peptide has a sequence having one or moresubstitutions from the group consisting of E6R, F26S, Y27L, V43L, F48T,R495, S50I, A54R, L55R, and K65R. In some embodiments, the vIGF2 peptidehas a sequence having a substitution of E6R. In some embodiments, thevIGF2 peptide has a sequence having a substitution of F26S. In someembodiments, the vIGF2 peptide has a sequence having a substitution ofY27L. In some embodiments, the vIGF2 peptide has a sequence having asubstitution of V43L. In some embodiments, the vIGF2 peptide has asequence having a substitution of F48T. In some embodiments, the vIGF2peptide has a sequence having a substitution of R495. In someembodiments, the vIGF2 peptide has a sequence having a substitution ofS50I. In some embodiments, the vIGF2 peptide has a sequence having asubstitution of A54R. In some embodiments, the vIGF2 peptide has asequence having a substitution of L55R. In some embodiments, the vIGF2peptide has a sequence having a substitution of K65R. In someembodiments, the vIGF2 peptide has a sequence having a substitution ofE6R, F26S, Y27L, V43L, F48T, R495, S50I, A54R, and L55R. In someembodiments, the vIGF2 peptide has an N-terminal deletion. In someembodiments, the vIGF2 peptide has an N-terminal deletion of one aminoacid. In some embodiments, the vIGF2 peptide has an N-terminal deletionof two amino acids. In some embodiments, the vIGF2 peptide has anN-terminal deletion of three amino acids. In some embodiments, the vIGF2peptide has an N-terminal deletion of four amino acids. In someembodiments, the vIGF2 peptide has an N-terminal deletion of four aminoacids and a substitution of E6R, Y27L, and K65R. In some embodiments,the vIGF2 peptide has an N-terminal deletion of four amino acids and asubstitution of E6R and Y27L. In some embodiments, the vIGF2 peptide hasan N-terminal deletion of five amino acids. In some embodiments, thevIGF2 peptide has an N-terminal deletion of six amino acids. In someembodiments, the vIGF2 peptide has an N-terminal deletion of seven aminoacids. In some embodiments, the vIGF2 peptide has an N-terminal deletionof seven amino acids and a substitution of Y27L and K65R.

TABLE 3 IGF2 Amino Acid Sequences (variant residues  are underlined) SEQID Peptide Sequence NO Wildtype AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASR  1VSRRSRGIVEECCFRSCDLALLETYCATPAKSE F26SAYRPSETLCGGELVDTLQFVCGDRGSYFSRPASR  2 VSRRSRGIVEECCFRSCDLALLETYCATPAKSEY27L AYRPSETLCGGELVDTLQFVCGDRGFLFSRPASRV  3SRRSRGIVEECCFRSCDLALLETYCATPAKSE V43L AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASR 4 VSRRSRGILEECCFRSCDLALLETYCATPAKSE F48TAYRPSETLCGGELVDTLQFVCGDRGFYFSRPASR  5 VSRRSRGIVEECCTRSCDLALLETYCATPAKSER49S AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASR  6VSRRSRGIVEECCFSSCDLALLETYCATPAKSE S50IAYRPSETLCGGELVDTLQFVCGDRGFYFSRPASR  7 VSRRSRGIVEECCFRICDLALLETYCATPAKSEA54R AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASR  8VSRRSRGIVEECCFRSCDLRLLETYCATPAKSE L55RAYRPSETLCGGELVDTLQFVCGDRGFYFSRPASR  9 VSRRSRGIVEECCFRSCDLARLETYCATPAKSEF26S,  AYRPSETLCGGELVDTLQFVCGDRGSLFSRPASRV 10 Y27L, SRRSRGILEECCTSICDLRRLETYCATPAKSE V43L, F48T,  R49S, S50I, A54R,  L55RΔ1-6,  TLCGGELVDTLQFVCGDRGFLFSRPASRVSRRSRG 11 Y27L, IVEECCFRSCDLALLETYCATPARSE K65R Δ1-7, LCGGELVDTLQFVCGDRGFLFSRPASRVSRRSRGI 30 Y27L,  VEECCFRSCDLALLETYCATPARSEK65R Δ1-4,  SRTLCGGELVDTLQFVCGDRGFLFSRPASRVSRRS 31 E6R, RGIVEECCFRSCDLALLETYCATPARSE Y27L, K65R Δ1-4, SRTLCGGELVDTLQFVCGDRGFLFSRPASRVSRRS 34 E6R, RGIVEECCFRSCDLALLETYCATPAKSE Y27L E6R AYRPSRTLCGGELVDTLQFVCGDRGFYFSRPASR35 VSRRSRGIVEECCFRSCDLALLETYCATPAKSE

TABLE 4 IGF2 DNA Coding Sequences SEQ  ID Peptide DNA Sequence NOMature  GCTTACCGCCCCAGTGAGACCCTGTGCGGCGGG 48 WT IGF2GAGCTGGTGGACACCCTCCAGTTCGTCTGTGGG GACCGCGGCTTCTACTTCAGCAGGCCCGCAAGCCGTGTGAGCCGTCGCAGCCGTGGCATCGTTGAG GAGTGCTGTTTCCGCAGCTGTGACCTGGCCCTCCTGGAGACGTACTGTGCTACCCCCGCCAAGTCC GAG vIGF2 TCTAGAACACTGTGCGGAGGGGAGCTTGTAGAC 36 Δ1-4, ACTCTTCAGTTCGTGTGTGGAGATCGCGGGTTC E6R,CTCTTCTCTCGCCCCGCTTCCAGAGTTTCACGGA Y27L, GGTCTAGGGGTATAGTAGAGGAGTGTTGTTTCA K65R GGTCCTGTGACTTGGCGCTCCTCGAGACCTATTGCGCGACGCCAGCCAGGTCCGAA

Internal Ribosomal Entry Sequences

Provided herein are gene therapy constructs useful in treating adisorder further comprising an internal ribosome entry sequence (IRES)for increasing gene expression by bypassing the bottleneck oftranslation initiation. Suitable internal ribosomal entry sequences foroptimizing expression for gene therapy include but are not limited to acricket paralysis virus (CrPV) IRES, a picornavirus IRES, an AphthovirusIRES, a Kaposi's sarcoma-associated herpesvirus IRES, a Hepatitis AIRES, a Hepatitis C IRES, a Pestivirus IRES, a Cripavirus IRES, aRhopalosiphum padi virus IRES, a Merek's disease virus IRES, and othersuitable IRES sequences. In some embodiments, the gene therapy constructcomprises a CrPV IRES. In some embodiments, the CrPV IRES has a nucleicacid sequence ofAAAAATGTGATCTTGCTTGTAAATACAATTTTGAGAGGTTAATAAATTACAAGTAGTGCTATTTTTGTATTTAGGTTAGCTATTTAGCTTTACGTTCCAGGATGCCTAGTGGCAGCCCCACAATATCCAGGAAGCCCTCTCTGCGGTTTTTCAGATTAGGTAGTCGAAAAACCTAAGAAATTTACCT GCT (SEQID NO: 12). In some embodiments, the CrPV IRES sequence is at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to SEQID NO: 12.

Signal Peptides

Gene therapy constructs provided herein, in some embodiments, furthercomprise a signal peptide, which improves secretion of the therapeuticprotein from the cell transduced with the gene therapy construct. Thesignal peptide in some embodiments improves protein processing oftherapeutic proteins, and facilitates translocation of the nascentpolypeptide-ribosome complex to the ER and ensuring properco-translational and post-translational modifications. In someembodiments, the signal peptide is located (i) in an upstream positionof the signal translation initiation sequence, (ii) in between thetranslation initiation sequence and the therapeutic protein, or (iii) adownstream position of the therapeutic protein. Signal peptides usefulin gene therapy constructs include but are not limited to bindingimmunoglobulin protein (BiP) signal peptide from the family of HSP70proteins (e.g., HSPA5, heat shock protein family A member 5) and Gaussiasignal peptides, and variants thereof. These signal peptides haveultrahigh affinity to the signal recognition particle. Examples of BiPand Gaussia amino acid sequences are provided in Table 5 below. In someembodiments, the signal peptide has an amino acid sequence that is atleast 90% identical to a sequence selected from the group consisting ofSEQ ID Nos: 13-17. In some embodiments, the signal peptide differs froma sequence selected from the group consisting of SEQ ID Nos: 13-17 by 5or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 amino acid.

TABLE 5 Signal Peptide Sequences   SEQ Signal ID  PeptideAmino Acid Sequence NO: Native  MKLSLVAAMLLLLSAARA 13 human BiPModified  MKLSLVAAMLLLLSLVAAMLLLLSAARA 14 BiP-1 Modified MKLSLVAAMLLLLWVALLLLSAARA 15 BiP-2 Modified  MKLSLVAAMLLLLSLVALLLLSAARA16 BiP-3 Modified  MKLSLVAAMLLLLALVALLLLSAARA 17 BiP-4 GaussiaMGVKVLFALICIAVAEA 32

The BiP signal peptide-signal recognition particle (SRP) interactionfacilitates translocation to the ER. This interaction is illustrated inFIG. 20 .

The Gaussia signal peptide is derived from the luciferase from Gaussiaprinceps and directs increased protein synthesis and secretion oftherapeutic proteins fused to this signal peptide. In some embodiments,the Gaussia signal peptide has an amino acid sequence that is at least90% identical to SEQ ID NO: 32. In some embodiments, the signal peptidediffers from SEQ ID NO: 32 by 5 or fewer, 4 or fewer, 3 or fewer, 2 orfewer, or 1 amino acid.

Linker

Gene therapy constructs provided herein, in some embodiments, comprise alinker between the targeting peptide and the therapeutic protein. Suchlinkers, in some embodiments, maintain correct spacing and mitigatesteric clash between the vIGF2 peptide and the therapeutic protein.Linkers, in some embodiments, comprise repeated glycine residues,repeated glycine-serine residues, and combinations thereof. In someembodiments, the linker consists of 5-20 amino acids, 5-15 amino acids,5-10 amino acids, 8-12 amino acids, or about 5, 6, 7, 8, 9, 10, 11, 12or 13 amino acids. Suitable linkers for gene therapy constructs hereininclude but are not limited to those provided in Table 6 below.

TABLE 6 Linker Sequences Sequence SEQ ID NO: GGGGSGGGG 18 GGGGS 19GGGSGGGGS 20 GGGGSGGGS 21 GGSGSGSTS 33 GGGGSGGGGS 37

Translation Initiation Sequence

Gene therapy constructs provided herein comprise a nucleic acid having atranslation initiation sequence, such as a Kozak sequence which aids ininitiation of translation of the mRNA. Kozak sequences contemplatedherein have a consensus sequence of (gcc)RccATGG (SEQ ID NO: 27) where alowercase letter denotes the most common base at the position and thebase varies, uppercase letters indicate highly conserved bases that onlyvary rarely change. R indicates that a purine (adenine or guanine) isalways observed at that position. The sequence in parentheses (gcc) isof uncertain significance. In some embodiments, the Kozak sequencecomprises the sequence AX₁X₂ATGA (SEQ ID NO: 28), wherein each of X₁ andX₂ is any nucleotide. In some embodiments, X₁ comprises A. In someembodiments, X₂ comprises G. In some embodiments, the Kozak sequencecomprises a nucleic acid sequence at least 85% identical to AAGATGA (SEQID NO: 29). In some embodiments, the Kozak sequence differs from thesequence of AAGATGA (SEQ ID NO: 29) by one or two nucleotides. In someembodiments, Kozak sequences provided herein have a sequence of AAGATGA(SEQ ID NO: 29). In some embodiments the Kozak sequence comprises anucleic acid sequence at least 85% identical to GCAAGATG (SEQ ID NO:44). In some embodiments the Kozak sequence differs from the sequence ofGCAAGATG (SEQ ID NO: 44) by one or two nucleotides. In some embodiments,the Kozak sequence comprises GCAAGATG (SEQ ID NO: 44). In someembodiments the Kozak sequence comprises a nucleic acid sequence atleast 85% identical to CACCATG (SEQ ID NO: 47). In some embodiments theKozak sequence differs from the sequence of CACCATG (SEQ ID NO: 47) byone or two nucleotides. In some embodiments, the Kozak sequencecomprises CACCATG (SEQ ID NO: 47).

Therapeutic Protein

Gene therapy constructs provided herein comprise a nucleic acid encodinga therapeutic protein for treating a genetic disorder due to a geneticdefect in an individual resulting in an absent or defective protein. Thetherapeutic protein expressed from the gene therapy construct replacesthe absent or defective protein. Therapeutic proteins, therefore, arechosen based on the genetic defect in need of treatment in anindividual. In some embodiments, the therapeutic protein is a structuralprotein. In some embodiments, the therapeutic protein is an enzyme. Insome embodiments, the therapeutic protein is a regulatory protein. Insome embodiments, the therapeutic protein is a receptor. In someembodiments, the therapeutic protein is a peptide hormone. In someembodiments, the therapeutic protein is a cytokine or a chemokine.

In some embodiments, gene therapy constructs herein encode an enzyme,such as an enzyme having a genetic defect in an individual with alysosomal storage disorder. In some embodiments, gene therapy constructsencode a lysosomal enzyme, such as a glycosidase, a protease, or asulfatase. In some embodiments, enzymes encoded by gene therapyconstructs provided herein include but are not limited toα-D-mannosidase; N-aspartyl-β-glucosaminidase; β-galactosidase;ceramidase; fucosidase; galactocerebrosidase; arylsulfatase A;N-acetylglucosamine-1-phosphotransferase; iduronate sulfatase;N-acetylglucosaminidase; acetyl-CoA:α-glucosaminide acetyltransferase;N-acetylglucosamine 6-sulfatase; β-glucuronidase; hyaluronidase;sialidase; sulfatase; sphingomyelinase; acid β-mannosidase; cathepsin K;3-hexosaminidase A; β-hexosaminidase B; α-N-acetylgalactosaminidase;sialin; hexosaminidase A; beta-glucosidase; α-iduronidase;α-galactosidase A; β-glucocerebrosidase; lysosomal acid lipase;glycosaminoglycan alpha-L-iduronohydrolase; iduronate-2-sulfatase;N-acetylgalactosamine-6-sulfatase; glycosaminoglycanN-acetylgalactosamine 4-sulfatase; alpha-glucosidase; heparansulfamidase; gp-91 subunit of NADPH oxidase; adenosine deaminase; cyclindependent kinase like 5; and palmitoyl protein thioesterase 1. In someembodiments, enzymes encoded by gene therapy constructs provided hereincomprise alpha-glucosidase. In some embodiments, the therapeutic proteinis associated with a genetic disorder selected from the group consistingof CDKL5 deficiency disorder, cystic fibrosis, alpha- andbeta-thalassemias, sickle cell anemia, Marfan syndrome, fragile Xsyndrome, Huntington's disease, hemochromatosis, Congenital Deafness(nonsyndromic), Tay-Sachs, Familial hypercholesterolemia, Duchennemuscular dystrophy, Stargardt disease, Usher syndrome, choroideremia,achromatopsia, X-linked retinoschisis, hemophilia, Wiskott-Aldrichsyndrome, X-linked chronic granulomatous disease, aromatic L-amino aciddecarboxylase deficiency, recessive dystrophic epidermolysis bullosa,alpha 1 antitrypsin deficiency, Hutchinson-Gilford progeria syndrome(HGPS), Noonan syndrome, X-linked severe combined immunodeficiency(X-SCID). In some embodiments, the therapeutic protein is selected fromthe group consisting of CDKL5, Connexin 26, hexosaminidase A, LDLreceptor, Dystrophin, CFTR, beta-globulin, HFE, Huntington, ABCA4,myosin VIIA (MYO7A), Rab escort protein-1 (REP1), cyclic nucleotidegated channel beta 3 (CNGB3), retinoschisin 1 (RS1), hemoglobin subunitbeta (HBB), Factor IX, WAS, cytochrome B-245 beta chain, dopadecarboxylase (DDC), collagen type VII alpha 1 chain (COL7A1), serpinfamily A member 1 (SERPINA1), LMNA, PTPN11, SOS1, RAF1, KRAS, and IL2receptor y gene.

Gene Therapy Vector Examples

Gene Therapy Vectors and Compositions

Provided herein are gene therapy vectors in which a nucleic acid, suchas a DNA, encoding a therapeutic fusion protein, such as a vIGF2 fusion,optionally having a signal peptide. The gene therapy vector optionallycomprises an internal ribosomal entry sequence. Vectors derived fromretroviruses such as the lentivirus are suitable tools to achievelong-term gene transfer since they allow long-term, stable integrationof a transgene and its propagation in daughter cells. Lentiviral andadeno-associated viral vectors have the added advantage over vectorsderived from onco-retroviruses such as murine leukemia viruses in thatthey are capable of transducing non-proliferating cells, such ashepatocytes and neurons. They also have the added advantage of lowimmunogenicity.

Exemplary gene therapy vectors herein encode therapeutic proteins andtherapeutic fusion proteins comprising a vIGF2 peptide. Nucleic acidsencoding exemplary fusion protein amino acid sequences are provided inTable 7 below.

TABLE 7 DNA Sequences SEQ ID  Construct DNA Sequence NO Kozak-GCAAGATGGGAGTGAGGCACCCGCCCTGCTCCCACCGGCTCCTGGCCG 44 hGAATCTGCGCCCTCGTGTCCTTGGCAACCGCTGCACTCCTGGGGCACATCCT (NaturalACTCCATGATTTCCTGCTGGTTCCCCGAGAGCTGAGTGGCTCCTCCCCA GAA)GTCCTGGAGGAGACTCACCCAGCTCACCAGCAGGGAGCCAGTAGACCAGGGCCCCGGGATGCCCAGGCACACCCCGGCCGTCCCAGAGCAGTGCCCACACAGTGCGACGTCCCCCCCAACAGCCGCTTCGATTGCGCCCCTGACAAGGCCATCACCCAGGAACAGTGCGAGGCCCGCGGCTGTTGCTACATCCCTGCAAAGCAGGGGCTGCAGGGAGCCCAGATGGGGCAGCCCTGGTGCTTCTTCCCACCCAGCTACCCCAGCTACAAGCTGGAGAACCTGAGCTCCTCTGAAATGGGCTACACGGCCACCCTGACCCGTACCACCCCCACCTTCTTCCCCAAGGACATCCTGACCCTGCGGCTGGACGTGATGATGGAGACTGAGAACCGCCTCCACTTCACGATCAAAGATCCAGCTAACAGGCGCTACGAGGTGCCCTTGGAGACCCCGCATGTCCACAGCCGGGCACCGTCCCCACTCTACAGCGTGGAGTTCTCCGAGGAGCCCTTCGGGGTGATCGTGCGCCGGCAGCTGGACGGCCGCGTGCTGCTGAACACGACGGTGGCGCCCCTGTTCTTTGCGGACCAGTTCCTTCAGCTGTCCACCTCGCTGCCCTCGCAGTATATCACAGGCCTCGCCGAGCACCTCAGTCCCCTGATGCTCAGCACCAGCTGGACCAGGATCACCCTGTGGAACCGGGACCTTGCGCCCACGCCCGGTGCGAACCTCTACGGGTCTCACCCTTTCTACCTGGCGCTGGAGGACGGCGGGTCGGCACACGGGGTGTTCCTGCTAAACAGCAATGCCATGGATGTGGTCCTGCAGCCGAGCCCTGCCCTTAGCTGGAGGTCGACAGGTGGGATCCTGGATGTCTACATCTTCCTGGGCCCAGAGCCCAAGAGCGTGGTGCAGCAGTACCTGGACGTTGTGGGATACCCGTTCATGCCGCCATACTGGGGCCTGGGCTTCCACCTGTGCCGCTGGGGCTACTCCTCCACCGCTATCACCCGCCAGGTGGTGGAGAACATGACCAGGGCCCACTTCCCCCTGGACGTCCAGTGGAACGACCTGGACTACATGGACTCCCGGAGGGACTTCACGTTCAACAAGGATGGCTTCCGGGACTTCCCGGCCATGGTGCAGGAGCTGCACCAGGGCGGCCGGCGCTACATGATGATCGTGGATCCTGCCATCAGCAGCTCGGGCCCTGCCGGGAGCTACAGGCCCTACGACGAGGGTCTGCGGAGGGGGGTTTTCATCACCAACGAGACCGGCCAGCCGCTGATTGGGAAGGTATGGCCCGGGTCCACTGCCTTCCCCGACTTCACCAACCCCACAGCCCTGGCCTGGTGGGAGGACATGGTGGCTGAGTTCCATGACCAGGTGCCCTTCGACGGCATGTGGATTGACATGAACGAGCCTTCCAACTTCATCAGGGGCTCTGAGGACGGCTGCCCCAACAATGAGCTGGAGAACCCACCCTACGTGCCTGGGGTGGTTGGGGGGACCCTCCAGGCGGCCACCATCTGTGCCTCCAGCCACCAGTTTCTCTCCACACACTACAACCTGCACAACCTCTACGGCCTGACCGAAGCCATCGCCTCCCACAGGGCGCTGGTGAAGGCTCGGGGGACACGCCCATTTGTGATCTCCCGCTCGACCTTTGCTGGCCACGGCCGATACGCCGGCCACTGGACGGGGGACGTGTGGAGCTCCTGGGAGCAGCTCGCCTCCTCCGTGCCAGAAATCCTGCAGTTTAACCTGCTGGGGGTGCCTCTGGTCGGGGCCGACGTCTGCGGCTTCCTGGGCAACACCTCAGAGGAGCTGTGTGTGCGCTGGACCCAGCTGGGGGCCTTCTACCCCTTCATGCGGAACCACAACAGCCTGCTCAGTCTGCCCCAGGAGCCGTACAGCTTCAGCGAGCCGGCCCAGCAGGCCATGAGGAAGGCCCTCACCCTGCGCTACGCACTCCTCCCCCACCTCTACACACTGTTCCACCAGGCCCACGTCGCGGGGGAGACCGTGGCCCGGCCCCTCTTCCTGGAGTTCCCCAAGGACTCTAGCACCTGGACTGTGGACCACCAGCTCCTGTGGGGGGAGGCCCTGCTCATCACCCCAGTGCTCCAGGCCGGGAAGGCCGAAGTGACTGGCTACTTCCCCTTGGGCACATGGTACGACCTGCAGACGGTGCCAGTAGAGGCCCTTGGCAGCCTCCCACCCCCACCTGCAGCTCCCCGTGAGCCAGCCATCCACAGCGAGGGGCAGTGGGTGACGCTGCCGGCCCCCCTGGACACCATCAACGTCCACCTCCGGGCTGGGTACATCATCCCCCTGCAGGGCCCTGGCCTCACAACCACAGAGTCCCGCCAGCAGCCCATGGCCCTGGCTGTGGCCCTGACCAAGGGTGGGGAGGCCCGAGGGGAGCTTTTCTGGGACGATGGAGAGAGCCTGGAAGTGCTGGAGCGAGGGGCCTACACACAGGTCATCTTCCTGGCCAGGAATAACACGATCGTGAATGAGCTGGTACGTGTGACCAGTGAGGGAGCTGGCCTGCAGCTGCAGAAGGTGACTGTCCTGGGCGTGGCCACGGCGCCCCAGCAGGTCCTCTCCAACGGTGTCCCTGTCTCCAACTTCACCTACAGCCCCGACACCAAGGTCCTGGACATCTGTGTCTCGCTGTTGATGGGAGAGCAGTTTCT CGTCAGCTGGTGTTAGKozak BiP- GCAAGATGAAGCTCTCCCTGGTGGCCGCGATGCTGCTGCTGCTCAGCG 38 vIGF2-GAACGGCGCGGGCCTCTAGAACACTGTGCGGAGGGGAGCTTGTAGACACTC (“EngineeredTTCAGTTCGTGTGTGGAGATCGCGGGTTCCTCTTCTCTCGCCCCGCTTCC hGAA”)AGAGTTTCACGGAGGTCTAGGGGTATAGTAGAGGAGTGTTGTTTCAGGTCCTGTGACTTGGCGCTCCTCGAGACCTATTGCGCGACGCCAGCCAGGTCCGAAGGGGGCGGTGGCTCAGGTGGTGGAGGTAGCAGACCAGGGCCCCGGGATGCCCAGGCACACCCCGGCCGTCCCAGAGCAGTGCCCACACAGTGCGACGTCCCCCCCAACAGCCGCTTCGATTGCGCCCCTGACAAGGCCATCACCCAGGAACAGTGCGAGGCCCGCGGCTGTTGCTACATCCCTGCAAAGCAGGGGCTGCAGGGAGCCCAGATGGGGCAGCCCTGGTGCTTCTTCCCACCCAGCTACCCCAGCTACAAGCTGGAGAACCTGAGCTCCTCTGAAATGGGCTACACGGCCACCCTGACCCGTACCACCCCCACCTTCTTCCCCAAGGACATCCTGACCCTGCGGCTGGACGTGATGATGGAGACTGAGAACCGCCTCCACTTCACGATCAAAGATCCAGCTAACAGGCGCTACGAGGTGCCCTTGGAGACCCCGCATGTCCACAGCCGGGCACCGTCCCCACTCTACAGCGTGGAGTTCTCCGAGGAGCCCTTCGGGGTGATCGTGCGCCGGCAGCTGGACGGCCGCGTGCTGCTGAACACGACGGTGGCGCCCCTGTTCTTTGCGGACCAGTTCCTTCAGCTGTCCACCTCGCTGCCCTCGCAGTATATCACNGGCCTCGCCGAGCACCTCAGTCCCCTGATGCTCAGCACCAGCTGGACCAGGATCACCCTGTGGAACCGGGACCTTGCGCCCACGCCCGGTGCGAACCTCTACGGGTCTCACCCTTTCTACCTGGCGCTGGAGGACGGCGGGTCGGCACACGGGGTGTTCCTGCTAAACAGCAATGCCATGGATGTGGTCCTGCAGCCGAGCCCTGCCCTTAGCTGGAGGTCGACAGGTGGGATCCTGGATGTCTACATCTTCCTGGGCCCAGAGCCCAAGAGCGTGGTGCAGCAGTACCTGGACGTTGTGGGATACCCGTTCATGCCGCCATACTGGGGCCTGGGCTTCCACCTGTGCCGCTGGGGCTACTCCTCCACCGCTATCACCCGCCAGGTGGTGGAGAACATGACCAGGGCCCACTTCCCCCTGGACGTCCAGTGGAACGACCTGGACTACATGGACTCCCGGAGGGACTTCACGTTCAACAAGGATGGCTTCCGGGACTTCCCGGCCATGGTGCAGGAGCTGCACCAGGGCGGCCGGCGCTACATGATGATCGTGGATCCTGCCATCAGCAGCTCGGGCCCTGCCGGGAGCTACAGGCCCTACGACGAGGGTCTGCGGAGGGGGGTTTTCATCACCAACGAGACCGGCCAGCCGCTGATTGGGAAGGTATGGCCCGGGTCCACTGCCTTCCCCGACTTCACCAACCCCACAGCCCTGGCCTGGTGGGAGGACATGGTGGCTGAGTTCCATGACCAGGTGCCCTTCGACGGCATGTGGATTGACATGAACGAGCCTTCCAACTTCATCAGGGGCTCTGAGGACGGCTGCCCCAACAATGAGCTGGAGAACCCACCCTACGTGCCTGGGGTGGTTGGGGGGACCCTCCAGGCGGCCACCATCTGTGCCTCCAGCCACCAGTTTCTCTCCACACACTACAACCTGCACAACCTCTACGGCCTGACCGAAGCCATCGCCTCCCACAGGGCGCTGGTGAAGGCTCGGGGGACACGCCCATTTGTGATCTCCCGCTCGACCTTTGCTGGCCACGGCCGATACGCCGGCCACTGGACGGGGGACGTGTGGAGCTCCTGGGAGCAGCTCGCCTCCTCCGTGCCAGAAATCCTGCAGTTTAACCTGCTGGGGGTGCCTCTGGTCGGGGCCGACGTCTGCGGCTTCCTGGGCAACACCTCAGAGGAGCTGTGTGTGCGCTGGACCCAGCTGGGGGCCTTCTACCCCTTCATGCGGAACCACAACAGCCTGCTCAGTCTGCCCCAGGAGCCGTACAGCTTCAGCGAGCCGGCCCAGCAGGCCATGAGGAAGGCCCTCACCCTGCGCTACGCACTCCTCCCCCACCTCTACACACTGTTCCACCAGGCCCACGTCGCGGGGGAGACCGTGGCCCGGCCCCTCTTCCTGGAGTTCCCCAAGGACTCTAGCACCTGGACTGTGGACCACCAGCTCCTGTGGGGGGAGGCCCTGCTCATCACCCCAGTGCTCCAGGCCGGGAAGGCCGAAGTGACTGGCTACTTCCCCTTGGGCACATGGTACGACCTGCAGACGGTGCCAGTAGAGGCCCTTGGCAGCCTCCCACCCCCACCTGCAGCTCCCCGTGAGCCAGCCATCCACAGCGAGGGGCAGTGGGTGACGCTGCCGGCCCCCCTGGACACCATCAACGTCCACCTCCGGGCTGGGTACATCATCCCCCTGCAGGGCCCTGGCCTCACAACCACAGAGTCCCGCCAGCAGCCCATGGCCCTGGCTGTGGCCCTGACCAAGGGTGGGGAGGCCCGAGGGGAGCTGTTCTGGGACGATGGAGAGAGCCTGGAAGTGCTGGAGCGAGGGGCCTACACACAGGTCATCTTCCTGGCCAGGAATAACACGATCGTGAATGAGCTGGTACGTGTGACCAGTGAGGGAGCTGGCCTGCAGCTGCAGAAGGTGACTGTCCTGGGCGTGGCCACGGCGCCCCAGCAGGTCCTCTCCAACGGTGTCCCTGTCTCCAACTTCACCTACAGCCCCGACACCAAGGTCCTGGACATCTGTGTCTCGCTGTTGATGGGAGAGCAGTTTCTCGTCAG CTGGTGTTAG CricketAAAAATGTGATCTTGCTTGTAAATACAATTTTGAGAGGTTAATAAATTACAAGT 39 ParalysisAGTGCTATTTTTGTATTTAGGTTAGCTATTTAGCTTTACGTTCCAGGATGCCTA Virus IRESGTGGCAGCCCCACAATATCCAGGAAGCCCTCTCTGCGGTTTTTCAGATTAGG (underlined)-TAGTCGAAAAACCTAAGAAATTTACCTGCT ATG AAGCTCTCCCTGGTGGCC BiP-vIGF2-GCGATGCTGCTGCTGCTCAGCGCGGCGCGGGCCTCTAGAACACTGTGC GAAGGAGGGGAGCTTGTAGACACTCTTCAGTTCGTGTGTGGAGATCGCGGGTTCCTCTTCTCTCGCCCCGCTTCCAGAGTTTCACGGAGGTCTAGGGGTATAGTAGAGGAGTGTTGTTTCAGGTCCTGTGACTTGGCGCTCCTCGAGACCTATTGCGCGACGCCAGCCAGGTCCGAAGGGGGCGGTGGCTCAGGTGGTGGAGGTAGCAGACCAGGGCCCCGGGATGCCCAGGCACACCCCGGCCGTCCCAGAGCAGTGCCCACACAGTGCGACGTCCCCCCCAACAGCCGCTTCGATTGCGCCCCTGACAAGGCCATCACCCAGGAACAGTGCGAGGCCCGCGGCTGTTGCTACATCCCTGCAAAGCAGGGGCTGCAGGGAGCCCAGATGGGGCAGCCCTGGTGCTTCTTCCCACCCAGCTACCCCAGCTACAAGCTGGAGAACCTGAGCTCCTCTGAAATGGGCTACACGGCCACCCTGACCCGTACCACCCCCACCTTCTTCCCCAAGGACATCCTGACCCTGCGGCTGGACGTGATGATGGAGACTGAGAACCGCCTCCACTTCACGATCAAAGATCCAGCTAACAGGCGCTACGAGGTGCCCTTGGAGACCCCGCATGTCCACAGCCGGGCACCGTCCCCACTCTACAGCGTGGAGTTCTCCGAGGAGCCCTTCGGGGTGATCGTGCGCCGGCAGCTGGACGGCCGCGTGCTGCTGAACACGACGGTGGCGCCCCTGTTCTTTGCGGACCAGTTCCTTCAGCTGTCCACCTCGCTGCCCTCGCAGTATATCACAGGCCTCGCCGAGCACCTCAGTCCCCTGATGCTCAGCACCAGCTGGACCAGGATCACCCTGTGGAACCGGGACCTTGCGCCCACGCCCGGTGCGAACCTCTACGGGTCTCACCCTTTCTACCTGGCGCTGGAGGACGGCGGGTCGGCACACGGGGTGTTCCTGCTAAACAGCAATGCCATGGATGTGGTCCTGCAGCCGAGCCCTGCCCTTAGCTGGAGGTCGACAGGTGGGATCCTGGATGTCTACATCTTCCTGGGCCCAGAGCCCAAGAGCGTGGTGCAGCAGTACCTGGACGTTGTGGGATACCCGTTCATGCCGCCATACTGGGGCCTGGGCTTCCACCTGTGCCGCTGGGGCTACTCCTCCACCGCTATCACCCGCCAGGTGGTGGAGAACATGACCAGGGCCCACTTCCCCCTGGACGTCCAGTGGAACGACCTGGACTACATGGACTCCCGGAGGGACTTCACGTTCAACAAGGATGGCTTCCGGGACTTCCCGGCCATGGTGCAGGAGCTGCACCAGGGCGGCCGGCGCTACATGATGATCGTGGATCCTGCCATCAGCAGCTCGGGCCCTGCCGGGAGCTACAGGCCCTACGACGAGGGTCTGCGGAGGGGGGTTTTCATCACCAACGAGACCGGCCAGCCGCTGATTGGGAAGGTATGGCCCGGGTCCACTGCCTTCCCCGACTTCACCAACCCCACAGCCCTGGCCTGGTGGGAGGACATGGTGGCTGAGTTCCATGACCAGGTGCCCTTCGACGGCATGTGGATTGACATGAACGAGCCTTCCAACTTCATCAGGGGCTCTGAGGACGGCTGCCCCAACAATGAGCTGGAGAACCCACCCTACGTGCCTGGGGTGGTTGGGGGGACCCTCCAGGCGGCCACCATCTGTGCCTCCAGCCACCAGTTTCTCTCCACACACTACAACCTGCACAACCTCTACGGCCTGACCGAAGCCATCGCCTCCCACAGGGCGCTGGTGAAGGCTCGGGGGACACGCCCATTTGTGATCTCCCGCTCGACCTTTGCTGGCCACGGCCGATACGCCGGCCACTGGACGGGGGACGTGTGGAGCTCCTGGGAGCAGCTCGCCTCCTCCGTGCCAGAAATCCTGCAGTTTAACCTGCTGGGGGTGCCTCTGGTCGGGGCCGACGTCTGCGGCTTCCTGGGCAACACCTCAGAGGAGCTGTGTGTGCGCTGGACCCAGCTGGGGGCCTTCTACCCCTTCATGCGGAACCACAACAGCCTGCTCAGTCTGCCCCAGGAGCCGTACAGCTTCAGCGAGCCGGCCCAGCAGGCCATGAGGAAGGCCCTCACCCTGCGCTACGCACTCCTCCCCCACCTCTACACACTGTTCCACCAGGCCCACGTCGCGGGGGAGACCGTGGCCCGGCCCCTCTTCCTGGAGTTCCCCAAGGACTCTAGCACCTGGACTGTGGACCACCAGCTCCTGTGGGGGGAGGCCCTGCTCATCACCCCAGTGCTCCAGGCCGGGAAGGCCGAAGTGACTGGCTACTTCCCCTTGGGCACATGGTACGACCTGCAGACGGTGCCAGTAGAGGCCCTTGGCAGCCTCCCACCCCCACCTGCAGCTCCCCGTGAGCCAGCCATCCACAGCGAGGGGCAGTGGGTGACGCTGCCGGCCCCCCTGGACACCATCAACGTCCACCTCCGGGCTGGGTACATCATCCCCCTGCAGGGCCCTGGCCTCACAACCACAGAGTCCCGCCAGCAGCCCATGGCCCTGGCTGTGGCCCTGACCAAGGGTGGGGAGGCCCGAGGGGAGCTGTTCTGGGACGATGGAGAGAGCCTGGAAGTGCTGGAGCGAGGGGCCTACACACAGGTCATCTTCCTGGCCAGGAATAACACGATCGTGAATGAGCTGGTACGTGTGACCAGTGAGGGAGCTGGCCTGCAGCTGCAGAAGGTGACTGTCCTGGGCGTGGCCACGGCGCCCCAGCAGGTCCTCTCCAACGGTGTCCCTGTCTCCAACTTCACCTACAGCCCCGACACCAAGGTCCTGGACATCTGTGTCTCGCTGTTGATGGGAGAGCAGTTTCTCGTCAGCTGGTGTTAG wt-PPT1ATGGCATCACCGGGTTGCCTCTGGTTGTTGGCCGTTGCGTTGCTTCCGT 40 IDT codonGGACATGTGCATCAAGAGCTCTTCAACATCTGGATCCCCCAGCTCCCCT optimizedGCCGCTCGTAATCTGGCACGGGATGGGGGATTCATGTTGTAACCCGTTGTCAATGGGCGCGATAAAAAAGATGGTTGAAAAGAAGATTCCAGGCATCTACGTTCTGTCCCTGGAAATCGGTAAGACACTGATGGAAGACGTGGAGAACTCCTTCTTTCTCAACGTCAATAGTCAGGTCACTACCGTCTGTCAAGCATTGGCAAAGGACCCTAAACTTCAGCAGGGGTACAATGCGATGGGGTTTAGCCAGGGCGGACAGTTTCTTAGAGCCGTCGCACAGCGCTGTCCATCTCCCCCGATGATTAACCTTATATCTGTCGGGGGACAACACCAGGGTGTTTTTGGTCTTCCTCGCTGTCCTGGTGAAAGCTCCCACATCTGTGATTTCATACGCAAAACGTTGAACGCAGGAGCTTATAGTAAAGTCGTCCAAGAACGGCTTGTTCAAGCGGAGTATTGGCATGACCCAATAAAAGAAGACGTTTATAGGAATCACTCTATCTTCTTGGCCGATATCAACCAAGAACGCGGAATCAACGAAAGCTACAAAAAGAATCTTATGGCTCTCAAGAAATTTGTTATGGTGAAATTCCTTAATGACTCTATAGTAGATCCTGTCGATTCAGAATGGTTCGGGTTCTACAGGTCTGGCCAGGCGAAGGAGACTATTCCCCTCCAAGAAACGTCTCTCTATACACAAGACAGACTCGGACTGAAAGAGATGGATAATGCGGGCCAGTTGGTCTTCTTGGCTACGGAAGGCGATCATCTCCAACTCTCCGAAGAGTGGTTCTATGCCCATATAATCCCGTTCCTGGGCTAA PPT1-2 (wt-ATGGCATCCCCCGGATGTTTGTGGCTGCTGGCGGTTGCGCTTCTGCCAT 41 vIGF2-PPT1;GGACGTGCGCCTCCCGAGCCCTCCAACACCTGTCCAGGACACTTTGCG CodonGCGGAGAGTTGGTCGATACGCTTCAATTCGTGTGTGGGGATAGAGGCT optimized byTCCTTTTTTCTCGGCCCGCTAGCCGCGTGTCCCGAAGGTCCCGGGGTAT IDT codonCGTTGAGGAATGCTGTTTCCGGTCCTGCGATCTTGCACTGTTGGAGACA optimizationTACTGTGCTACGCCTGCGAGAAGCGAGGGTGGAGGGGGTTCTGGAGGT tool)GGAGGGAGCCGGCCTCGGGCGGTTCCCACCCAGGATCCTCCAGCTCCTCTGCCTCTGGTCATCTGGCATGGGATGGGGGACTCATGTTGTAACCCGCTGAGTATGGGGGCAATTAAAAAAATGGTTGAAAAGAAAATTCCAGGTATTTATGTCCTCTCTCTTGAAATCGGTAAGACACTTATGGAGGATGTGGAAAACTCCTTTTTCCTTAATGTCAATTCTCAGGTCACAACAGTTTGTCAGGCTCTGGCGAAGGATCCTAAGCTGCAGCAAGGCTACAACGCCATGGGTTTTTCCCAGGGAGGCCAATTTCTCAGAGCGGTAGCTCAGCGATGTCCATCACCACCGATGATAAATCTGATCAGTGTCGGCGGACAACACCAGGGAGTTTTCGGGCTGCCCAGGTGTCCGGGGGAATCTAGTCACATATGTGACTTCATTCGCAAGACCCTTAACGCCGGCGCTTACTCAAAGGTGGTTCAAGAACGGCTTGTGCAGGCTGAATACTGGCACGATCCCATCAAGGAAGATGTATATAGGAACCACAGTATCTTTCTGGCAGACATAAATCAGGAAAGGGGTATTAACGAAAGCTACAAGAAAAATCTCATGGCCCTGAAGAAATTTGTAATGGTTAAGTTTTTGAACGATTCTATAGTAGATCCTGTTGACTCCGAGTGGTTCGGGTTCTATCGATCTGGTCAAGCCAAGGAGACGATTCCGCTTCAGGAAACTTCACTGTACACACAGGATCGGCTGGGACTCAAGGAGATGGACAATGCGGGCCAGTTGGTGTTTCTGGCTACAGAGGGAGACCATCTCCAGTTGAGTGAAGAATGGTTCTATGCACATATTATCCCATTCCTCGGCTA A PPT1-29ATGAAGCTCTCCCTGGTGGCCGCGATGCTGCTGCTGCTCTGGGTGGCAC 42 (BiP2aa-TGCTGCTGCTCAGCGCGGCGAGGGCCGCCGCGAGTCGCACGTTGTGTG vIGF2-PPT1;GAGGTGAACTCGTCGACACCCTTCAGTTCGTATGTGGAGATCGCGGTTT native humanCCTCTTCTCACGCCCAGCTTCCAGAGTTTCCCGAAGATCACGAGGAATA sequence)GTTGAGGAGTGCTGTTTTCGGTCTTGTGATCTGGCTCTCCTCGAGACTTATTGTGCTACGCCGGCCCGCTCTGAAGGAGGTGGTGGCAGTGGAGGAGGAGGGAGTCGGCCTAGGGCAGTCCCAACCCAGGACCCGCCGGCGCCGCTGCCGTTGGTGATCTGGCATGGGATGGGAGACAGCTGTTGCAATCCCTTAAGCATGGGTGCTATTAAAAAAATGGTGGAGAAGAAAATACCTGGAATTTACGTCTTATCTTTAGAGATTGGGAAGACCCTGATGGAGGACGTGGAGAACAGCTTCTTCTTGAATGTCAATTCCCAAGTAACAACAGTGTGTCAGGCACTTGCTAAGGATCCTAAATTGCAGCAAGGCTACAATGCTATGGGATTCTCCCAGGGAGGCCAATTTCTGAGGGCAGTGGCTCAGAGATGCCCTTCACCTCCCATGATCAATCTGATCTCGGTTGGGGGACAACATCAAGGTGTTTTTGGACTCCCTCGATGCCCAGGAGAGAGCTCTCACATCTGTGACTTCATCCGAAAAACACTGAATGCTGGGGCGTACTCCAAAGTTGTTCAGGAACGCCTCGTGCAAGCCGAATACTGGCATGACCCCATAAAGGAGGATGTGTATCGCAACCACAGCATCTTCTTGGCAGATATAAATCAGGAGCGGGGTATCAATGAGTCCTACAAGAAAAACCTGATGGCCCTGAAGAAGTTTGTGATGGTGAAATTCCTCAATGATTCCATTGTGGACCCTGTAGATTCGGAGTGGTTTGGATTTTACAGAAGTGGCCAAGCCAAGGAAACCATTCCCTTACAGGAGACCTCCCTGTACACACAGGACCGCCTGGGGCTAAAGGAAATGGACAATGCAGGACAGCTAGTGTTTCTGGCTACAGAAGGGGACCATCTTCAGTTGTCTGAAGAATGGTTTTATGCCCACATCATACCATTCCTTGGATG A

In some embodiments, the vector comprising the nucleic acid encoding thedesired therapeutic fusion protein, such as a vIGF2 fusion or a signalpeptide fusion, optionally having an internal ribosomal entry sequence,provided herein is an adeno-associated viral vector (A5/35).

In some embodiments, the nucleic acid encoding the therapeutic fusionprotein, such as a vIGF2 fusion, optionally having an internal ribosomalentry sequence, is cloned into a number of types of vectors. Forexample, in some embodiments, the nucleic acid is cloned into a vectorincluding, but not limited to a plasmid, a phagemid, a phage derivative,an animal virus, and a cosmid. Vectors of particular interest includeexpression vectors, replication vectors, probe generation vectors, andsequencing vectors.

Further, the expression vector encoding the therapeutic fusion protein,such as a vIGF2 fusion or a signal peptide fusion, optionally having aninternal ribosomal entry sequence, in some embodiments, is provided to acell in the form of a viral vector. Viral vector technology isdescribed, e.g., in Sambrook et al., 2012, Molecular Cloning: ALaboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY), and inother virology and molecular biology manuals. Viruses, which are usefulas vectors include, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendonuclease sites, and one or more selectable markers (e.g., WO01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

Also provided herein are compositions and systems for gene transfer. Anumber of virally based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. A selected gene, in someembodiments, is inserted into a vector and packaged in retroviralparticles using suitable techniques. The recombinant virus is thenisolated and delivered to cells of the subject either in vivo or exvivo. A number of retroviral systems are suitable for gene therapy. Insome embodiments, adenovirus vectors are used. A number of adenovirusvectors are suitable for gene therapy. In some embodiments,adeno-associated virus vectors are used. A number of adeno-associatedviruses are suitable for gene therapy. In one embodiment, lentivirusvectors are used.

Gene therapy constructs provided herein comprise a vector (or genetherapy expression vector) into which the gene of interest is cloned orotherwise which includes the gene of interest in a manner such that thenucleotide sequences of the vector allow for the expression(constitutive or otherwise regulated in some manner) of the gene ofinterest. The vector constructs provided herein include any suitablegene expression vector that is capable of being delivered to a tissue ofinterest and which will provide for the expression of the gene ofinterest in the selected tissue of interest.

In some embodiments, the vector is an adeno-associated virus (AAV)vector because of the capacity of AAV vectors to cross the blood-brainbarrier and transduction of neuronal tissue. In methods provided herein,AAV of any serotype is contemplated to be used. The serotype of theviral vector used in certain embodiments is selected from the groupconsisting of an AAV1 vector, an AAV2 vector, an AAV3 vector, an AAV4vector, an AAV5 vector, an AAV6 vector, an AAV7 vector, an AAV8 vector,an AAV9 vector, an AAVrhS vector, an AAVrh10 vector, an AAVrh33 vector,an AAVrh34 vector, an AAVrh74 vector, an AAV Anc80 vector, an AAVPHP.Bvector, an AAVhu68 vector, an AAV-DJ vector, and others suitable forgene therapy.

AAV vectors are DNA parvoviruses that are nonpathogenic for mammals.Briefly, AAV-based vectors have the rep and cap viral genes that accountfor 96% of the viral genome removed, leaving the two flanking 145 basepair inverted terminal repeats (ITRs) which are used to initiate viralDNA replication, packaging, and integration.

Further embodiments include use of other serotype capsids to create anAAV1 vector, an AAV2 vector, an AAV3 vector, an AAV4 vector, an AAV5vector, an AAV6 vector, an AAV7 vector, an AAV8 vector, an AAV9 vector,an AAVrhS vector, an AAVrh10 vector, an AAVrh33 vector, an AAVrh34vector, an AAVrh74 vector, an AAV Anc80 vector, an AAVPHP.B vector, anAAV-DJ vector, and others suitable for gene therapy. Optionally, the AAVviral capsid is AAV2/9, AAV9, AAVrhS, AAVrh10, AAVAnc80, or AAV PHP.B.

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave been shown to contain functional elements downstream of the startsite as well. The spacing between promoter elements frequently isflexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements is often increased to 50bp apart before activity begins to decline. Depending on the promoter,it appears that individual elements function either cooperatively orindependently to activate transcription.

An example of a promoter that is capable of expressing a therapeuticfusion protein, such as a vIGF2 fusion or a signal peptide fusion,optionally having an internal ribosomal entry sequence, transgene in amammalian T-cell is the EF1a promoter. The native EF1a promoter drivesexpression of the alpha subunit of the elongation factor-1 complex,which is responsible for the enzymatic delivery of aminoacyl tRNAs tothe ribosome. The EF1a promoter has been extensively used in mammalianexpression plasmids and has been shown to be effective in drivingexpression from transgenes cloned into a lentiviral vector (see, e.g.,Milone et al., Mol. Ther. 17(8): 1453-1464 (2009)). Another example of apromoter is the immediate early cytomegalovirus (CMV) promoter sequence.This promoter sequence is a strong constitutive promoter sequencecapable of driving high levels of expression of any polynucleotidesequence operatively linked thereto. However, other constitutivepromoter sequences are sometimes also used, including, but not limitedto the chicken β actin promoter, the P546 promoter, the simian virus 40(SV40) early promoter, mouse mammary tumor virus (MMTV), humanimmunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLVpromoter, an avian leukemia virus promoter, an Epstein-Barr virusimmediate early promoter, a Rous sarcoma virus promoter, as well ashuman gene promoters such as, but not limited to, the actin promoter,the myosin promoter, the elongation factor-1a promoter, the hemoglobinpromoter, and the creatine kinase promoter. Further, gene therapyvectors are not contemplated to be limited to the use of constitutivepromoters. Inducible promoters are also contemplated here. The use of aninducible promoter provides a molecular switch capable of turning onexpression of the polynucleotide sequence which it is operatively linkedwhen such expression is desired, or turning off the expression whenexpression is not desired. Examples of inducible promoters include, butare not limited to a metallothionine promoter, a glucocorticoidpromoter, a progesterone promoter, and a tetracycline-regulatedpromoter.

In order to assess the expression of a therapeutic fusion protein, suchas a vIGF fusion or a signal peptide fusion, optionally having aninternal ribosomal entry sequence, or portions thereof, the expressionvector to be introduced into a cell often contains either a selectablemarker gene or a reporter gene or both to facilitate identification andselection of expressing cells from the population of cells sought to betransfected or infected through viral vectors. In other aspects, theselectable marker is often carried on a separate piece of DNA and usedin a co-transfection procedure. Both selectable markers and reportergenes are sometimes flanked with appropriate regulatory sequences toenable expression in the host cells. Useful selectable markers include,for example, antibiotic-resistance genes, such as neo and the like.

Methods and compositions for introducing and expressing genes into acell are suitable for methods herein. In the context of an expressionvector, the vector is readily introduced into a host cell, e.g.,mammalian, bacterial, yeast, or insect cell by any method in the art.For example, the expression vector is transferred into a host cell byphysical, chemical, or biological means.

Physical methods and compositions for introducing a polynucleotide intoa host cell include calcium phosphate precipitation, lipofection,particle bombardment, microinjection, gene gun, electroporation, and thelike. Methods for producing cells comprising vectors and/or exogenousnucleic acids are suitable for methods herein (see, e.g., Sambrook etal., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, ColdSpring Harbor Press, NY). One method for the introduction of apolynucleotide into a host cell is calcium phosphate transfection.

Chemical means and compositions for introducing a polynucleotide into ahost cell include colloidal dispersion systems, such as macromoleculecomplexes, nanocapsules, microspheres, beads, and lipid-based systemsincluding oil-in-water emulsions, micelles, mixed micelles, nucleicacid-lipid particles, and liposomes. An exemplary colloidal system foruse as a delivery vehicle in vitro and in vivo is a liposome (e.g., anartificial membrane vesicle). Other methods of state-of-the-art targeteddelivery of nucleic acids are available, such as delivery ofpolynucleotides with targeted nanoparticles or other suitable sub-micronsized delivery system.

In the case where a non-viral delivery system is utilized, an exemplarydelivery vehicle is a liposome. The use of lipid formulations iscontemplated for the introduction of the nucleic acids into a host cell(in vitro, ex vivo or in vivo). In another aspect, the nucleic acid isassociated with a lipid. The nucleic acid associated with a lipid, insome embodiments, is encapsulated in the aqueous interior of a liposome,interspersed within the lipid bilayer of a liposome, attached to aliposome via a linking molecule that is associated with both theliposome and the oligonucleotide, entrapped in a liposome, complexedwith a liposome, dispersed in a solution containing a lipid, mixed witha lipid, combined with a lipid, contained as a suspension in a lipid,contained or complexed with a micelle, or otherwise associated with alipid. Lipid, lipid/DNA or lipid/expression vector associatedcompositions are not limited to any particular structure in solution.For example, in some embodiments, they are present in a bilayerstructure, as micelles, or with a “collapsed” structure. Alternately,they are simply be interspersed in a solution, possibly formingaggregates that are not uniform in size or shape. Lipids are fattysubstances which are, in some embodiments, naturally occurring orsynthetic lipids. For example, lipids include the fatty droplets thatnaturally occur in the cytoplasm as well as the class of compounds whichcontain long-chain aliphatic hydrocarbons and their derivatives, such asfatty acids, alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use are obtained from commercial sources. Forexample, in some embodiments, dimyristyl phosphatidylcholine (“DMPC”) isobtained from Sigma, St. Louis, Mo.; in some embodiments, dicetylphosphate (“DCP”) is obtained from K & K Laboratories (Plainview, N.Y.);cholesterol (“Choi”), in some embodiments, is obtained fromCalbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and otherlipids are often obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolare often stored at about −20° C. Chloroform is used as the only solventsince it is more readily evaporated than methanol. “Liposome” is ageneric term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes are often characterized as having vesicularstructures with a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh et al.,1991 Glycobiology 5: 505-10). However, compositions that have differentstructures in solution than the normal vesicular structure are alsoencompassed. For example, the lipids, in some embodiments, assume amicellar structure or merely exist as non-uniform aggregates of lipidmolecules. Also contemplated are lipofectamine-nucleic acid complexes.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise expose a cell to the therapeutic fusionprotein, such as a vIGF2 fusion or a signal peptide fusion, optionallyhaving an internal ribosomal entry sequence, provided herein, in orderto confirm the presence of the recombinant DNA sequence in the hostcell, a variety of assays are contemplated to be performed. Such assaysinclude, for example, “molecular biological” assays suitable for methodsherein, such as Southern and Northern blotting, RT-PCR and PCR;“biochemical” assays, such as detecting the presence or absence of aparticular peptide, e.g., by immunological means (ELISAs and westernblots) or by assays described herein to identify agents falling withinthe scope herein.

The present disclosure further provides a vector comprising atherapeutic fusion protein, such as a vIGF2 fusion or a signal peptidefusion, optionally having an internal ribosomal entry sequence, encodingnucleic acid molecule. In one aspect, a therapeutic fusion proteinvector is capable of being directly transduced into a cell. In oneaspect, the vector is a cloning or expression vector, e.g., a vectorincluding, but not limited to, one or more plasmids (e.g., expressionplasmids, cloning vectors, minicircles, minivectors, double minutechromosomes), retroviral and lentiviral vector constructs. In oneaspect, the vector is capable of expressing the vIGF2-therapeutic fusionprotein construct in mammalian cells. In one aspect, the mammalian cellis a human cell.

Uses and Methods of Treatment

Also provided herein are methods of treating genetic disorders usinggene therapy comprising administering to an individual a nucleic acidencoding a therapeutic fusion protein (such as a vIGF2 fusion or asignal peptide fusion or a signal peptide-vIGF2 fusion), optionallyhaving an internal ribosomal entry sequence, disclosed herein. Geneticdisorders suitable for treatment using methods herein comprise disordersin an individual caused by one or more mutations in the genome causinglack of expression or expression of a dysfunctional protein by themutant gene.

Further provided herein are pharmaceutical compositions comprising agene therapy vector, such as a gene therapy vector comprising a nucleicacid encoding a therapeutic fusion protein (such as a vIGF2 fusion or asignal peptide fusion or a signal peptide-vIGF2 fusion), optionallyhaving an internal ribosomal entry sequence, disclosed herein and apharmaceutically acceptable carrier or excipient for use in preparationof a medicament for treatment of a genetic disorder.

Genetic disorders suitable for treatment by methods herein include butare not limited to Achondroplasia, Alpha-1 Antitrypsin Deficiency,Antiphospholipid Syndrome, Autosomal Dominant Polycystic Kidney Disease,Charcot-Marie-Tooth, Colon cancer, Cri du chat, Crohn's Disease, Cysticfibrosis, Dercum Disease, Duane Syndrome, Duchenne Muscular Dystrophy,Factor V Leiden Thrombophilia, Familial Hypercholesterolemia, FamilialMediterranean Fever, Fragile X Syndrome, Gaucher Disease,Hemochromatosis, Hemophilia, Holoprosencephaly, Huntington's disease,Klinefelter syndrome, Marfan syndrome, Myotonic Dystrophy,Neurofibromatosis, Noonan Syndrome, Osteogenesis Imperfecta, Parkinson'sdisease, Phenylketonuria, Poland Anomaly, Porphyria, Progeria, RetinitisPigmentosa, Severe Combined Immunodeficiency (SCID), Sickle celldisease, Spinal Muscular Atrophy, Tay-Sachs disease, Thalassemia,Trimethylaminuria, Turner Syndrome, Velocardiofacial Syndrome, WAGRSyndrome, or Wilson Disease. In some embodiments, the genetic disorderis selected from the group consisting of CDKL5 deficiency disorder,cystic fibrosis, alpha- and beta-thalassemias, sickle cell anemia,Marfan syndrome, fragile X syndrome, Huntington's disease,hemochromatosis, Congenital Deafness (nonsyndromic), Tay-Sachs, Familialhypercholesterolemia, Duchenne muscular dystrophy, Stargardt disease,Usher syndrome, choroideremia, achromatopsia, X-linked retinoschisis,hemophilia, Wiskott-Aldrich syndrome, X-linked chronic granulomatousdisease, aromatic L-amino acid decarboxylase deficiency, recessivedystrophic epidermolysis bullosa, alpha 1 antitrypsin deficiency,Hutchinson-Gilford progeria syndrome (HGPS), Noonan syndrome, X-linkedsevere combined immunodeficiency (X-SCID).

In some embodiments, genetic disorders suitable for treatment usingmethods provided herein are lysosomal storage disorder. In someembodiments, lysosomal storage disorders are treated herein using genetherapy to deliver missing or defective enzymes to the patient. In someembodiments, methods herein deliver an enzyme fused to a vIGF2 or fusedto a signal peptide to the patient in order to deliver the enzyme to thecell where it is needed. In some embodiments, the lysosomal storagedisorders is selected from the group consisting ofaspartylglucosaminuria, Batten disease, cystinosis, Fabry disease,Gaucher disease type I, Gaucher disease type II, Gaucher disease typeIII, Pompe disease, Tay Sachs disease, Sandhoff disease, metachomaticleukodystrophy, mucolipidosis type I, mucolipidosis type II,mucolipidosis type III, mucolipidosis type IV, Hurler disease, Hunterdisease, Sanfilippo disease type A, Sanfilippo disease type B,Sanfilippo disease type C, Sanfilippo disease type D, Morquio diseasetype A, Morquio disease type B, Maroteau-Lamy disease, Sly disease,Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pickdisease type C1, Niemann-Pick disease type C2, Schindler disease type I,and Schindler disease type II. In some embodiments, the lysosomalstorage disorder is selected from the group consisting of activatordeficiency, GM2-gangliosidosis; GM2-gangliosidosis, AB variant;alpha-mannosidosis (type 2, moderate form; type 3, neonatal, severe);beta-mannosidosis; aspartylglucosaminuria; lysosomal acid lipasedeficiency; cystinosis (late-onset juvenile or adolescent nephropathictype; infantile nephropathic); Chanarin-Dorfman syndrome; neutral lipidstorage disease with myopathy; NLSDM; Danon disease; Fabry disease;Fabry disease type II, late-onset; Farber disease; Farberlipogranulomatosis; fucosidosis; galactosialidosis (combinedneuraminidase & beta-galactosidase deficiency); Gaucher disease; type IIGaucher disease; type III Gaucher disease; type IIIC Gaucher disease;Gaucher disease, atypical, due to saposin C deficiency;GM1-gangliosidosis (late-infantile/juvenile GM1-gangliosidosis;adult/chronic GM1-gangliosidosis); Globoid cell leukodystrophy, Krabbedisease (Late infantile onset; Juvenile Onset; Adult Onset); Krabbedisease, atypical, due to saposin A deficiency; MetachromaticLeukodystrophy (juvenile; adult); partial cerebroside sulfatedeficiency; pseudoarylsulfatase A deficiency; metachromaticleukodystrophy due to saposin B deficiency; Mucopolysaccharidosesdisorders: MPS I, Hurler syndrome; MPS I, Hurler-Scheie syndrome; MPS I,Scheie syndrome; MPS II, Hunter syndrome; MPS II, Hunter syndrome;Sanfilippo syndrome Type A/MPS IIIA; Sanfilippo syndrome Type B/MPSIIIB; Sanfilippo syndrome Type C/MPS IIIC; Sanfilippo syndrome TypeD/MPS IIID; Morquio syndrome, type A/MPS IVA; Morquio syndrome, typeB/MPS IVB; MPS IX hyaluronidase deficiency; MPS VI Maroteaux-Lamysyndrome; MPS VII Sly syndrome; mucolipidosis I, sialidosis type II;I-cell disease, Leroy disease, mucolipidosis II; Pseudo-Hurlerpolydystrophy/mucolipidosis type III; mucolipidosis IIIC/ML III GAMMA;mucolipidosis type IV; multiple sulfatase deficiency; Niemann-Pickdisease (type B; type C1/chronic neuronopathic form; type C2; typeD/Nova Scotian type); Neuronal Ceroid Lipofuscinoses: CLN6disease—Atypical Late Infantile, Late-Onset variant, Early Juvenile;Batten-Spielmeyer-Vogt/Juvenile NCL/CLN3 disease; Finnish Variant LateInfantile CLN5; Jansky-Bielschowsky disease/Late infantile CLN2/TPP1Disease; Kufs/Adult-onset NCL/CLN4 disease (type B); NorthernEpilepsy/variant late infantile CLN8; Santavuori-Haltia/InfantileCLN1/PPT disease; Pompe disease (glycogen storage disease type II);late-onset Pompe disease; Pycnodysostosis; Sandhoff disease/GM2gangliosidosis; Sandhoff disease/GM2 gangliosidosis; Sandhoffdisease/GM2 Gangliosidosis; Schindler disease (type III/intermediate,variable); Kanzaki disease; Salla disease; infantile free sialic acidstorage disease (ISSD); spinal muscular atrophy with progressivemyoclonic epilepsy (SMAPME); Tay-Sachs disease/GM2 gangliosidosis;juvenile-onset Tay-Sachs disease; late-onset Tay-Sachs disease;Christianson syndrome; Lowe oculocerebrorenal syndrome;Charcot-Marie-Tooth type 4J, CMT4J; Yunis-Varon syndrome; bilateraltemporooccipital polymicrogyria (BTOP); X-linked hypercalciuricnephrolithiasis, Dent-1; and Dent disease 2. In some embodiments, thetherapeutic protein is associated with a lysosomal storage disorder andthe therapeutic protein is selected from the group consisting ofGM2-activator protein; α-mannosidase; MAN2B1; lysosomal β-mannosidase;glycosylasparaginase; lysosomal acid lipase; cystinosin; CTNS; PNPLA2;lysosome-associated membrane protein-2; α-galactosidase A; GLA; acidceramidase; α-L-fucosidase; protective protein/cathepsin A; acidβ-glucosidase; GBA; PSAP; β-galactosidase-1; GLB1; galactosylceramideβ-galactosidase; GALC; PSAP; arylsulfatase A; ARSA; α-L-iduronidase;iduronate 2-sulfatase; heparan N-sulfatase; N-α-acetylglucosaminidase;heparan acetyl CoA: α-glucosaminide acetyltransferase;N-acetylglucosamine 6-sulfatase; galactosamine-6-sulfate sulfatase;β-galactosidase; hyaluronidase; arylsulfatase B; β-glucuronidase;neuraminidase; NEU1; gamma subunit ofN-acetylglucosamine-1-phosphotransferase; mucolipin-1;sulfatase-modifying factor-1; acid sphingomyelinase; SMPD1; NPC1; andNPC2.

In some embodiments, treatment via methods herein delivers a geneencoding a therapeutic protein to a cell in need of the therapeuticprotein. In some embodiments, the treatment delivers the gene to allsomatic cells in the individual. In some embodiments, the treatmentreplaces the defective gene in the targeted cells. In some embodiments,cells treated ex vivo to express the therapeutic protein are deliveredto the individual.

Gene therapy for disorders disclosed herein provides superior treatmentoutcomes to conventional treatments, including enzyme replacementtherapy, because it does not require long infusion treatments. Inaddition, it has reduced risk of the individual developing an immuneresponse to the therapeutic protein, which is often experienced inindividuals receiving enzyme replacement therapy.

Definitions

As used herein “ex vivo gene therapy” refers to methods where patientcells are genetically modified outside the subject, for example toexpress a therapeutic gene. Cells with the new genetic information arethen returned to the subject from whom they were derived.

As used herein “in vivo gene therapy” refers to methods where a vectorcarrying the therapeutic gene(s) is directly administered to thesubject.

As used herein “fusion protein” and “therapeutic fusion protein” areused interchangeably herein and refer to a therapeutic protein having atleast one additional protein, peptide, or polypeptide, linked to it. Insome instances, fusion proteins are a single protein molecule containingtwo or more proteins or fragments thereof, covalently linked via peptidebond within their respective peptide chains, without chemical linkers.In some embodiments, the fusion protein comprises a therapeutic proteinand a signal peptide, a peptide that increases endocytosis of the fusionprotein, or both. In some embodiments, the peptide that increasesendocytosis is a peptide that binds CI-MPR.

As used herein “vector”, or “gene therapy vector”, used interchangeablyherein, refers to gene therapy delivery vehicles, or carriers, thatdeliver therapeutic genes to cells. A gene therapy vector is any vectorsuitable for use in gene therapy, e.g., any vector suitable for thetherapeutic delivery of nucleic acid polymers (encoding a polypeptide ora variant thereof) into target cells (e.g., sensory neurons) of apatient. In some embodiments, the gene therapy vector delivers thenucleic acid encoding a therapeutic protein or therapeutic fusionprotein to a cell where the therapeutic protein or fusion is expressedand secreted from the cell. The vector may be of any type, for exampleit may be a plasmid vector or a minicircle DNA. Typically, the vector isa viral vector. These include both genetically disabled viruses such asadenovirus and nonviral vectors such as liposomes. The viral vector mayfor example be derived from an adeno-associated virus (AAV), aretrovirus, a lentivirus, a herpes simplex virus, or an adenovirus. AAVderived vectors. The vector may comprise an AAV genome or a derivativethereof.

“Construct” as used herein refers to a nucleic acid molecule or sequencethat encodes a therapeutic protein or fusion protein and optionallycomprises additional sequences such as a translation initiation sequenceor IRES sequence.

As used herein “plasmid” refers to circular, double-stranded unit of DNAthat replicates within a cell independently of the chromosomal DNA.

As used herein “promoter” refers to a site on DNA to which the enzymeRNA polymerase binds and initiates the transcription of DNA into RNA.

As used herein “somatic therapy” refers to methods where themanipulation of gene expression in cells that will be corrective to thepatient but not inherited by the next generation. Somatic cells includeall the non-reproductive cells in the human body

As used herein “somatic cells” refers to all body cells except thereproductive cells.

As used herein “tropism” refers to preference of a vector, such as avirus for a certain cell or tissue type. Various factors determine theability of a vector to infect a particular cell. Viruses, for example,must bind to specific cell surface receptors to enter a cell. Virusesare typically unable to infect a cell if it does not express thenecessary receptors.

The term “transduction” is used to refer to the administration/deliveryof the nucleic acid encoding the therapeutic protein to a target celleither in vivo or in vitro, via a replication-deficient rAAV of thedisclosure resulting in expression of a functional polypeptide by therecipient cell. Transduction of cells with a gene therapy vector such asa rAAV of the disclosure results in sustained expression of polypeptideor RNA encoded by the rAAV. The present disclosure thus provides methodsof administering/delivering to a subject a gene therapy vector such asan rAAV encoding a therapeutic protein by an intrathecal, intraretinal,intraocular, intravitreous, intracerebroventricular, intraparechymal, orintravenous route, or any combination thereof “Intrathecal” deliveryrefers to delivery into the space under the arachnoid membrane of thebrain or spinal cord. In some embodiments, intrathecal administration isvia intracisternal administration.

The terms “recipient”, “individual”, “subject”, “host”, and “patient”,are used interchangeably herein and in some cases, refer to anymammalian subject for whom diagnosis, treatment, or therapy is desired,particularly humans. “Mammal” for purposes of treatment refers to anyanimal classified as a mammal, including humans, domestic and farmanimals, and laboratory, zoo, sports, or pet animals, such as dogs,horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guineapigs, monkeys etc. In some embodiments, the mammal is human.

As used herein, the terms “treatment,” “treating,” “ameliorating asymptom,” and the like, in some cases, refer to administering an agent,or carrying out a procedure, for the purposes of obtaining a therapeuticeffect, including inhibiting, attenuating, reducing, preventing oraltering at least one aspect or marker of a disorder, in a statisticallysignificant manner or in a clinically significant manner. The term“ameliorate” or “treat” does not state or imply a cure for theunderlying condition. “Treatment,” or “to ameliorate” (and like) as usedherein, may include treating a mammal, particularly in a human, andincludes: (a) preventing the disorder or a symptom of a disorder fromoccurring in a subject which may be predisposed to the disorder but hasnot yet been diagnosed as having it (e.g., including disorders that maybe associated with or caused by a primary disorder; (b) inhibiting thedisorder, i.e., arresting its development; (c) relieving the disorder,i.e., causing regression of the disorder; and (d) improving at least onesymptom of the disorder. Treating may refer to any indicia of success inthe treatment or amelioration or prevention of a disorder, including anyobjective or subjective parameter such as abatement; remission;diminishing of symptoms or making the disorder condition more tolerableto the patient; slowing in the rate of degeneration or decline; ormaking the final point of degeneration less debilitating. The treatmentor amelioration of symptoms is based on one or more objective orsubjective parameters; including the results of an examination by aphysician. Accordingly, the term “treating” includes the administrationof the compounds or agents of the present invention to prevent or delay,to alleviate, or to arrest or inhibit development of the symptoms orconditions associated with the disorder. The term “therapeutic effect”refers to the reduction, elimination, or prevention of the disorder,symptoms of the disorder, or side effects of the disorder in thesubject.

The term “affinity” refers to the strength of binding between a moleculeand its binding partner or receptor.

As used herein, the phrase “high affinity” refers to, for example, atherapeutic fusion containing such a peptide that binds CI-MPR which hasan affinity to CI-MPR that is about 100 to 1,000 times or 500 to 1,000times higher than that of the therapeutic protein without the peptide.In some embodiments, the affinity is at least 100, at least 500, or atleast 1000 times higher than without the peptide. For example, where thetherapeutic protein and CI-MPR are combined in relatively equalconcentration, the peptide of high affinity will bind to the availableCI-MPR so as to shift the equilibrium toward high concentration of theresulting complex.

“Secretion” as used herein refers to the release of a protein from acell into, for example, the bloodstream to be carried to a tissue ofinterest or a site of action of the therapeutic protein. When a genetherapy product is secreted into the interstitial space of an organ,secretion can allow for cross-correction of neighboring cells.

“Delivery” as used herein means drug delivery. In some embodiments, theprocess of delivery means transporting a drug substance (e.g.,therapeutic protein or fusion protein produced from a gene therapyvector) from outside of a cell (e.g., blood, tissue, or interstitialspace) into a target cell for therapeutic activity of the drugsubstance.

“Engineering” or “protein engineering” as used here in refers to themanipulation of the structures of a protein by providing appropriate anucleic acid sequence that encodes for the protein as to produce desiredproperties, or the synthesis of the protein with particular structures.

A “therapeutically effective amount” in some cases means the amountthat, when administered to a subject for treating a disorder, issufficient to effect treatment for that disorder.

As used herein, the term “about” a number refers to a range spanningthat from 10% less than that number through 10% more than that number,and including values within the range such as the number itself.

As used herein, the term “comprising” an element or elements of a claimrefers to those elements but does not preclude the inclusion of anadditional element or elements.

EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. The present examples, along with the methodsdescribed herein are presently representative of preferred embodiments,are exemplary, and are not intended as limitations on the scope of theinvention. Changes therein and other uses which are encompassed withinthe spirit of the invention as defined by the scope of the claims willoccur to those skilled in the art.

Example 1: Binding of Variant IGF2 Peptide to CI-MPRReceptor

Surface plasmon resonance (SPR) experiments were conducted using Biacoreto measure binding of wildtype and variant IGF2 (vIGF2) to the CI-MPRreceptor. The wildtype, human mature IGF2 peptide (wt IGF2) has thesequence set forth in SEQ ID NO: 1. The vIGF2 sequence differs from wtIGF2 in that it lacks residues 1-4 and contains the following mutations:E6R, Y27L, and K65R. It has the amino sequence:SRTLCGGELVDTLQFVCGDRGFLFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPARSE (SEQ IDNO: 31). vIGF2 also has an N-terminal linker with the sequence GGGGSGGGG(SEQ ID NO: 18). The combined sequence isGGGGSGGGGSRTLCGGELVDTLQFVCGDRGFLFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPARSE SEQ ID NO: 43). FIG. 4 shows that as expected, the wildtype IGF2peptide binds to the CI-MPRreceptor with high affinity (0.2 nM). FIG. 5shows that the variant IGF2 peptide (vIGF2) also binds to theCI-MPRreceptor with high affinity (0.5 nM). These data indicate thatvIGF2 peptide has high affinity for the intended CI-MPRreceptor fortargeting therapeutics to lysosomes.

SPR was utilized to measure peptide binding to the Insulin Receptor toassess potential side effects. Insulin binds the Insulin Receptor withhigh affinity (˜8 nM; data not shown). Wildtype IGF2 and a vIGF2 weretested, where the vIGF2 had the sequenceSRTLCGGELVDTLQFVCGDRGFLFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPARSE (SEQ IDNO: 31) having an N-terminal linker with a sequence GGGGSGGGG (SEQ IDNO: 18). FIG. 8 shows that wildtype IGF2 also binds the Insulin Receptorwith relatively high affinity (˜100 nM). IGF2 peptide fromBiomarin/Zystor IGF2-GAA fusion protein (BMN-701) also binds the InsulinReceptor with high affinity and was shown to cause hypoglycemia inclinical trials. FIG. 9 shows no measurable binding of vIGF2 peptide tothe insulin receptor. These data show that vIGF2 peptide confers asuperior safety profile compared with wt IGF2 peptide fusions.

The same SPR binding analysis was utilized to characterize vIGF2 peptideinteraction with the IGF1 Receptor. FIG. 10 shows that the wildtype IGF2peptide binds IGF1 receptor with relatively high affinity (˜100 nM).FIG. 11 shows no measurable binding of vIGF2 peptide to the IGF1Receptor, showing an improved safety profile compared to wt IGF2.

TABLE 8 SPR Affinity Results Receptor wt IGF2 Kd (nM) vIGF2 Kd (nM)CI-MPR 0.2 0.5 Insulin Receptor 100 No Binding Detected IGF1 Receptor100 No Binding Detected

Example 2: vIGF2 Converts Low Affinity Ligand to High Affinity ERT forCI-MPR

The vIGF2 peptide (SEQ ID NO: 31) with an N-terminal linker (SEQ ID NO:18) was chemically coupled to alglucosidase-alfa, designated here asvIGF2-alglucosidase-alfa, to determine whether the vIGF2 peptide couldimprove affinity for CI-MPR. As shown in FIG. 6 , binding affinities ofalglucosidase-alfa and vIGF2-alglucosidase-alfa were directly comparedusing CI-MPR plate binding assays in 96-well ELISA plates coated withCI-MPR. Unbound enzyme was washed away prior to measuring bound enzymeactivity. Varying concentrations of both enzyme preparations were usedwith or without free WT IGF2 peptide. vIGF2 substantially improved theaffinity for CI-MPR. Further, binding of vIGF2-alglucosidase-alfa wasblocked by free WT IGF2 indicating that binding was IGF2-dependent.(Data not shown.) Coupling of vIGF2 peptide did not impair GAA enzymeactivity.

The vIGF2 was coupled to recombinant human N-acetyl-α-D-glucosaminidase(rhNAGLU). RrhNAGLU, a lysosomal enzyme lacking M6P, to determinewhether peptide can convert a non-ligand to high affinity ligand forCI-MPR. In this experiment, rhNAGLU and vIGF2-rhNAGLU were directlycompared using CI-MPRplate binding assays, utilizing CI-MPR-coatedplates. Unbound enzyme was washed away prior to measuring bound enzymeactivity. Varying concentrations of both enzyme preparations were usedwith or without free vIGF2 peptide. As shown in FIG. 7 , vIGF2-rhNAGLUhas significantly higher affinity for CI-MPR than rhNAGLU lacking vIGF2.Further, vIGF2-rhNAGLU binding was blocked by free vIGF2 peptideindicating that receptor binding was specific for IGF2 peptide. Theseresults show that vIGF2 peptide can be utilized to improve drugtargeting to lysosomes.

Example 3: Myoblast Uptake of vIGF2-GAA Fusion Proteins

vIGF2-GAA fusion proteins (same sequences as in Examples 1-2) wereadministered and L6 myoblast uptake of the enzyme was measured. FIG. 6shows superior uptake of the vIGF2-rhGAA compared to rhGAA and M6P-GAA.Therefore, vIGF2 is effective at targeting GAA to the cells.

Example 4: Constructs for ERT Delivered by Gene Therapy

Two different constructs are illustrated in FIG. 12 . In the top panelis a construct which contains a Kozak sequence and a nucleic acidencoding a recombinant human GAA with the native signal peptide (SEQ IDNO: 45), encoding “natural hGAA” (SEQ ID NO: 45). In the middle panel isthe construct Kozak-BiP-vIGF2-2GS-GAA, encoding “engineered hGAA” (SEQID NO: 23). This construct is characterized by a Kozak sequence, anucleic acid encoding BiP signal peptide, a nucleic acid encoding thevIGF2 peptide having the sequence set forth in SEQ ID NO: 31, and anucleic acid encoding a 2GS linker (SEQ ID NO:18) followed by a nucleicacid encoding a recombinant human GAA with the N-terminal 60 amino acidsremoved (SEQ ID NO:46) to prevent premature processing and removal ofthe vIGF2.

Example 5: Enhanced Secretion of Gene Therapy Constructs

Engineered hGAA has greater secretion and is able to interact with acell surface receptor appropriate for cellular uptake and lysosomaltargeting

CHO expressing engineered hGAA, described in more detail below, ornatural hGAA were cultured and conditioned media was collected formeasurement of GAA activity. FIG. 15 shows the relative activity ofengineered and natural hGAA showing that engineered hGAA has increasedactivity compared to natural hGAA, indicative of more effecicientsecretion of engineered hGAA.

Example 6: Analysis of PPT1 in Conditioned Media

Cloning of PPT1 Constructs

PPT1 constructs were cloned into the pcDNA3.1 expression vector(ThermoFisher cat #V79020), which contains a CMV promoter. The testedconstructs included PPT1-1 (WT-PPT1) (SEQ ID NO: 24); PPT1-2(WT-vIGF2-PPT1) (SEQ ID NO: 25); PPT1-29 (BiP2aa-vIGF2-PPT1) (SEQ ID NO:26).

PPT1 Secretion & Binding

The PPT1 constructs were transiently expressed in HEK293T cells for 3days and the PPT1 secreted into the media. Secreted PPT1 was quantifiedby Western Blotting, and assayed for CI-MPR binding using establishedmethods. Secreted PPT1 is shown in FIG. 13 . CI-MPR binding is shown inFIG. 14 .

Example 7: Testing Gene Therapy Vectors in an Animal Model of PompeDisease

Pompe Gene Therapy: Preclinical Proof of Concept Study Design

A preclinical study was conducted in GAA knockout (GAA KO) mice using ahigh dose for initial comparison of constructs. The constructs are shownin FIG. 12 . Mice were treated with vehicle or one of two constructs,Natural—hGAA or Engineered—hGAA. Mice were administered 5e11 gc/mouse(approximately 2.5e13 gc/kg). GAA knockout mice were used at age 2months. Normal (wildtype) mice were used as a control. The study designis outlined in FIG. 16 .

Pompe Gene Therapy: Plasma

Plasma was collected from wild type (normal) mice or GAA KO mice treatedwith vehicle or a gene therapy vector as indicated and GAA activity andcell surface binding was measured. Data are summarized in FIG. 17 , FIG.27 , and FIG. 19 . Similar high GAA levels were seen in mice treatedwith gene therapy vectors (FIG. 17 , FIG. 18 ). However, greater celltargeting receptor binding was observed with the engineered construct(FIG. 19 ).

Pompe Gene Therapy: Quadriceps

GAA activity, and glycogen storage/cytoplasmic vacuolization wereassessed in normal (wild type) mice and treated GAA KO mice (FIG. 28 ).GAA activity in the quadriceps was about 20 fold higher than wild type.Glycogen PAS (FIG. 29 ) and immunohistochemistry (FIG. 30 ) were alsoassessed. Immunohistochemistry showed greater lysosomal targeting ofengineered hGAA compared to wild type. Glycogen reduction was moreconsistent for engineered hGAA by PAS staining.

Pompe Gene Therapy: Triceps

GAA activity, and glycogen storage/cytoplasmic vacuolization wereassessed in normal (wild type) mice and in treated GAA KO mice (FIG. 31). GAA activity was about 10-15 fold higher than wild type.Immunohistochemistry and glycogen PAS were also assessed (FIG. 32 andFIG. 33 ). Immunohistochemistry illustrated greater lysosomal targetingof engineered hGAA compared to wildtype GAA. Glycogen reduction was moreconsistent for engineered hGAA as measured by PAS staining.

Pompe Gene Therapy: Tibialis Anterior (TA)

GAA activity, and glycogen storage/cytoplasmic vacuolization wereassessed in normal (wild type) and treated GAA KO mice (FIG. 20 ). GAAactivity in the TA was about 15-20 fold higher than wild type.Immunohistochemistry and glycogen PAS were also assessed (FIG. 21 andFIG. 22 ). Immunohistochemistry illustrated greater lysosomal targetingof engineered hGAA compared to wildtype GAA. Glycogen levels were closeto wildtype levels. Glycogen reduction was more consistent forengineered hGAA by PAS staining.

Pompe Gene Therapy: Brain and Spinal Cord

GAA activity, glycogen content, and glycogen storage/cytoplasmicvacuolization were assessed in normal (wild type) mice and treated GAAKO mice (FIG. 23 ). GAA activity in the brain was about 5 fold lowerthan wildtype. Immunohistochemistry and glycogen PAS were also assessed(FIG. 24 , FIG. 25 , FIG. 26 , FIG. 27 ). Immunohistochemistry indicatedthat there may be a direct transduction of some cells. However, littleto no glycogen clearance was obtained with the natural construct.Glycogen levels were close to wild type levels for the engineeredconstruct even though activity was only 20% of wild type. PAS stainingin the spinal cord shows little to no glycogen clearance with thenatural construct. Glycogen levels close to wild type for engineeredconstruct was observed in the ventral horn including motor neurons.Immunohistochemistry demonstrated direct transduction in spinal cordneurons. Engineered hGAA produced by the choroid plexus and neuronalcells was able to reduce glycogen by cross correction in the spinal cordwhile little glycogen reduction was observed for natural hGAA.

Conclusions

Overall the data in this example demonstrated that the engineered genetherapy constructs have dramatically better uptake into tissues andglycogen reduction than the wildtype GAA used in conventionaltreatments, including effects in the brain and spinal cord.

Example 8: Animal Study Protocols

AAVhu68 vectors were produced and titrated by the Penn Vector Core asdescribed. (Lock, Alvira et al. 2010, “Rapid, simple, and versatilemanufacturing of recombinant adeno-associated viral vectors at scale.”Hum Gene Ther 21(10): 1259-1271).

Mus musculus, Pompe mice Gaa knock-out, in a C57BL/6/129 backgroundfounders were purchased at Jackson Labs (stock #004154, also known as6neo mice).

Mice received 5×10¹¹ GCs (approximately 2.5×10¹³ GC/kg) ofAAVhu68.CAG.hGAA (comprising either natural hGAA (SEQ ID NO: 45) orengineered hGAA (SEQ ID NO: 38) in 0.1 mL via the lateral tail vein,were bled on Day 7 and Day 21 post vector dosing for serum isolation,and were terminally bled (for plasma isolation) and euthanized byexsanguination 28 days post injection. Tissues were promptly collected,starting with brain.

GAA Activity

Plasma was mixed with 5.6 mM 4-MU-α-glucopyranoside pH 4.0 and incubatedfor three hours at 37° C. The reaction was stopped with 0.4 M sodiumcarbonate, pH 11.5. Relative fluorescence units, RFUs were measuredusing a Victor3 fluorimeter, ex 355 nm and emission at 460 nm. Activityin units of nmol/mL/hr was calculated by interpolation from a standardcurve of 4-MU. Activity in individual tissue samples were furthernormalized based on total protein content in the homogenate.

GAA Signature Peptide by LC/MS

Plasma was precipitated in 100% methanol and centrifuged. Supernatantswere discarded. The pellet was spiked with a stable isotope-labeledpeptide unique to hGAA as an internal standard and resuspended withtrypsin and incubated at 37° C. for one hour. The digestion was stoppedwith 10% formic acid. Tryptic peptides were separated by C-18 reversephase chromatography and Identified and quantified by ESI-massspectroscopy. The total GAA concentration in plasma was calculated fromthe signature peptide concentration.

Cell Surface Receptor Binding Assay

A 96-well plate was coated with receptor, washed, and blocked with BSA.28 day plasma from AAV treated mice was serially diluted to give aseries of decreasing concentrations and incubated with coupled receptor.After incubation the plate was washed to remove any unbound hGAA and4-MU-α-glucopyranoside added for one hour at 37° C. The reaction wasstopped with 1.0 M glycine, pH 10.5 and RFUs were read by a Spectramaxfluorimeter; ex 370, emission 460. RFU's for each sample were convertedto activity (nmol/mL/hr) by interpolation from a standard curve of 4-MU.Nonlinear regression was done using GraphPad Prism.

Histology

Tissues were formalin fixed and paraffin embedded. Muscle slides werestained with PAS; CNS slides with luxol fast blue/Periodic Acid-Schiff(PAS). A board certified veterinary pathologist (JH) blindly reviewedhistological slides. A semi-quantitative estimation of the totalpercentage of cells with glycogen storage and cytoplasmic vacuolizationwas done on scanned slides. A score from 0 to 4 was attributed asdescribed in table below.

TABLE 9 Histology Scoring Storage/Vacuolization 0 0 1 1 to 9% 2 10 to49% 3 50 to 74% 4 75 to 100%

Immuno-Histochemistry (IHC)

We studied transgene expression and cellular localization from slidesimmunostained using an anti-human GAA antibody (Sigma HPA029126).

Example 9: Histology-Tissue Processing-Protocols and Results in anAnimal Model of Pompe Disease

All tissues were fixed in 10% NBF (neutral buffered formalin). Theassays (PAS and IHC) are routinely used in the field.

PAS staining of quadriceps and triceps (FIG. 29 and FIG. 32 )—Tissueswere fixed in 10% NBF and embedded in paraffin. Sections were post-fixedin 1% periodic acid and stained with Schiff's reagent. Afterwards,sections were counterstained with hematoxylin. Glycogen appears asmagenta aggregates (lysosomal bound) or diffused pink (cytosolic);nuclei are blue. Based on the images and assuming each is representativeof a group, the ranking order in terms of glycogen clearance is:Engineered hGAA >Natural hGAA. The Engineered hGAA construct producedmore staining across the entire image compared to the rest, showing animproved endocytosis of GAA protein mediated through the binding ofvIGF2 to CI-MPR.

PAS staining of spinal cord (FIG. 26 )—Tissues were fixed in 10% NBF.Post-fixation in 1% periodic acid could have been done prior to or afterparaffin embedding. Sections were stained with Schiff's reagent andcounterstained likely with methylene blue. Glycogen appears as magentaaggregates (lysosomal bound); nerve fibers appear blue. The imagesfocused on the ventral horn of the spinal cord and glycogen accumulationin the motor neurons. Engineered hGAA appeared most effective inglycogen reduction among the constructs.

GAA IHC (FIG. 22 , FIG. 25 , FIG. 27 , FIG. 30 , and FIG. 35 )—Tissueswere fixed in 10% NBF and embedded in paraffin. Sections were incubatedwith an anti-GAA primary antibody, followed by a secondary antibody thatrecognizes the primary antibody and carries an enzyme tag—HRP.Subsequently, an enzymatic reaction was carried out and a brown-coloredprecipitating product was formed. Sections were then counterstained withhematoxylin. The constructs showed GAA uptake into muscle fibers (FIG.31 ). Engineered hGAA >Natural hGAA. The BiP-vIGF2 construct had morediffused staining across the entire image compared to the rest.

Compared to other vectors, engineered hGAA produced more GAA IHC signalswith a punctum-like appearance inside the muscle fibers, showing a muchmore efficient lysosomal targeting (FIG. 22 ).

In all, engineered hGAA consistently demonstrated superiority in tissueuptake, lysosomal targeting, and glycogen reduction in various tissuesamong the constructs.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments described herein may beemployed. It is intended that the following claims define the scope ofthe invention and that methods and structures within the scope of theseclaims and their equivalents be covered thereby.

What is claimed is:
 1. A gene therapy vector comprising a nucleic acidconstruct encoding a polypeptide comprising: (a) a therapeutic protein;(b) a peptide that binds to the cation-independent mannose 6-phosphate(M6P) receptor (CI-MPR) with high affinity; and (c) a linker between thetherapeutic protein and the peptide that binds CI-MPR.
 2. The genetherapy vector of claim 1, wherein the peptide is a variant IGF2 (vIGF2)peptide.
 3. The gene therapy vector of claim 2, wherein the vIGF2peptide comprises an amino acid sequence that is at least 90% identicalto SEQ ID NO: 1 and having at least one substitution at one or morepositions selected from the group consisting of positions 6, 26, 27, 43,48, 49, 50, 54, 55, and 65 of SEQ ID NO:
 1. 4. The gene therapy vectorof claim 3, wherein the at least one substitution is selected from thegroup consisting of E6R, F26S, Y27L, V43L, F48T, R495, S50I, A54R, L55R,and K65R of SEQ ID NO:1.
 5. The gene therapy vector of claim 3, whereinthe vIGF2 peptide comprises at least two substitutions at two or morepositions selected from the group consisting of positions 6, 26, 27, 43,48, 49, 50, 54, 55, 65 of SEQ ID NO:
 1. 6. The gene therapy vector ofclaim 5, wherein the at least two substitutions are selected from thegroup consisting of E6R, F26S, Y27L, V43L, F48T, R495, S50I, A54R, L55R,K65R of SEQ ID NO:
 1. 7. The gene therapy vector of claim 1, wherein thevIGF2 peptide comprises an N-terminal deletion at positions 1-4 of SEQID NO:
 1. 8. The gene therapy vector of claim 1, wherein the vIGF2peptide has decreased affinity for insulin receptor and IGF1R ascompared to native IGF2 peptide.
 9. The gene therapy vector of claim 2,wherein the vIGF2 peptide is capable of facilitating uptake of thetherapeutic protein into a cell.
 10. The gene therapy vector of claim 2,wherein the vIGF2 peptide is capable of facilitating uptake of thetherapeutic protein into a lysosome.
 11. The gene therapy vector ofclaim 1, wherein the therapeutic protein is capable of replacing adefective or deficient protein associated with a genetic disorder in asubject having the genetic disorder.
 12. The gene therapy vector ofclaim 11, wherein the genetic disorder is a lysosomal storage disorder.13. The gene therapy vector of claim 11, wherein the genetic disorder isselected from the group consisting of aspartylglucosaminuria, Battendisease, cystinosis, Fabry disease, Gaucher disease type I, Gaucherdisease type II, Gaucher disease type III, Pompe disease, Tay Sachsdisease, Sandhoff disease, metachomatic leukodystrophy, mucolipidosistype I, mucolipidosis type II, mucolipidosis type III, mucolipidosistype IV, Hurler disease, Hunter disease, Sanfilippo disease type A,Sanfilippo disease type B, Sanfilippo disease type C, Sanfilippo diseasetype D, Morquio disease type A, Morquio disease type B, Maroteau-Lamydisease, Sly disease, Niemann-Pick disease type A, Niemann-Pick diseasetype B, Niemann-Pick disease type C1, Niemann-Pick disease type C2,Schindler disease type I, Schindler disease type II, adenosine deaminasesevere combined immunodeficiency (ADA-SCID), chronic granulomatousdisease (CGD), and neuronal ceroid lipofuscinosis.
 14. The gene therapyvector of claim 11, wherein the genetic disorder is Pompe disease. 15.The gene therapy vector of claim 11, wherein the genetic disorder is aCLN1 disease.
 16. The gene therapy vector of claim 1, wherein thetherapeutic protein comprises a soluble lysosomal enzyme or anenzymatically active fragment thereof.
 17. The gene therapy vector ofclaim 1, wherein the therapeutic protein comprises a lysosomal enzyme oran enzymatically active fragment thereof, wherein the lysosomal enzymeis selected from the group consisting of alpha-galactosidase A,β-glucocerebrosidase, glucocerebrosidase, lysosomal acid lipase,glycosaminoglycan alpha-L-iduronohydrolase, iduronate-2-sulfatase,N-acetylgalactosamine-6-sulfatase, glycosaminoglycanN-acetylgalactosamine 4-sulfatase, palmitoyl protein thioesterases,cyclin dependent kinase like 5, and alpha-glucosidase.
 18. The genetherapy vector of claim 17, wherein the therapeutic protein isalpha-glucosidase or an enzymatically active fragment thereof.
 19. Thegene therapy vector of claim 17, wherein the therapeutic protein ispalmitoyl protein thioesterase-1 or an enzymatically active fragmentthereof.
 20. The gene therapy vector of claim 1, wherein the nucleicacid construct further comprises a translation initiation sequence. 21.The gene therapy vector of claim 1, wherein the nucleic acid constructfurther comprises a nucleic acid sequence encoding a signal peptidewherein the signal peptide is capable of increasing secretion of thetherapeutic protein as compared to the therapeutic protein without thesignal peptide.
 22. The gene therapy vector of claim 21, wherein thesignal peptide is selected from a binding immunoglobulin protein (BiP)signal peptide and a Gaussia signal peptide.
 23. The gene therapy vectorof claim 1, wherein the vIGF2 peptide comprises the sequence of SEQ IDNO:31.
 24. The gene therapy vector of claim 1, wherein the constructcomprises SEQ ID NO:36.
 25. The gene therapy vector of claim 1, whereinthe polypeptide comprises SEQ ID NO:23.
 26. The gene therapy vector ofclaim 1, wherein the construct comprises SEQ ID NO:38.
 27. The genetherapy vector of claim 1, wherein the vIGF2 at the N-terminus of thepolypeptide.
 28. The gene therapy vector of claim 1, wherein the vIGF2is at the C-terminus of the polypeptide.
 29. The gene therapy vector ofclaim 1, wherein the linker peptide comprises SEQ ID NO: 18-21 or SEQ IDNO:
 33. 30. The gene therapy vector of claim 1, wherein the gene therapyvector is a virus vector selected from the group consisting of anadenovirus vector, an adeno-associated virus (AAV) vector, a retrovirusvector, a lentivirus vector, a pox virus vector, a vaccinia virusvector, an adenovirus vector, and a herpes virus vector.